JP5113853B2 - Antibody specifically binding to Aβ oligomer and use thereof - Google Patents

Description

  The present invention relates to an antibody that specifically binds to an Aβ oligomer and use thereof.

  Various evidences indicate that memory loss is due to synaptic dysfunction caused by soluble Aβ oligomers (see Non-Patent Documents 1 and 2). Excessive accumulation and deposition of Aβ oligomers may trigger a series of pathological cascades leading to Alzheimer's disease (AD). Therefore, therapeutic intervention targeting Aβ oligomers may be effective in blocking this pathological cascade. However, the knowledge about the neurodegeneration mediated by the core responsible molecule, especially Aβ oligomers, is the core of this amyloid cascade hypothesis, which was born mainly from in vitro experiments (see Non-Patent Document 3) and directly proved in vivo. It has not been. The biggest drawback of the in vivo experiments reported so far (see Non-patent Document 4) is that the synaptic toxic effects of endogenous Aβ polymers have not been elucidated due to the lack of conformation-specific molecular tools. is there. There is no way in the human brain to solve a difficult situation even in Alzheimer's disease model mice, and the in vivo neurotoxicity of endogenous Aβ has often been ignored. It remains unclear why NFT formation and neuronal loss precede senile plaque formation and how Aβ oligomers are involved in the human entorhinal cortex.

  The prior art document information related to the present invention includes the following.

Klein WL, Trends Neurosci 24: 219-224, 2001. Selkoe DJ, Science 298: 789-791, 2002. Hass C et al: Nature Reviw 8: 101-12, 2007. Lee EB, et al: J Biol Chem. 281: 4292-4299, 2006.

  This invention is made | formed in view of the said situation, and makes it a subject to provide the antibody couple | bonded specifically with A (beta) oligomer, and its use. More specifically, an antibody that specifically binds to an Aβ oligomer, a method for measuring an Aβ oligomer using the antibody, a method for diagnosing Alzheimer's disease using the antibody, and a drug containing the antibody are provided. Objective.

The present inventors did not recognize the soluble amyloid β (Aβ) monomer, which is a physiological molecule, but produced a monoclonal antibody specific only for the soluble Aβ oligomer, and confirmed the following five functions:
(1) anti-neurotoxic activity;
(2) Aβ amyloid fibril formation inhibitory activity;
(3) Specificity that recognizes only Aβ oligomers;
(4) Ability to capture Aβ oligomers in AD brain;
(5) Ability to prevent the onset of Alzheimer's disease-like phenotype (memory impairment, brain Aβ accumulation level) in APPswe-transgenic mice (Tg2576).

  Among the antibodies produced, monoclonal 1A9 or 2C3 does not recognize monomers (approximately 4.5 kDa), but specifically recognizes oligomers of 30 kDa or more, mainly 100 kDa or more, as determined by ultrafiltration and molecular sieving. It was confirmed. Next, as a result of examining the neurotoxicity neutralizing effect induced by Aβ1-42 in PC12 cells differentiated into nerve cells, it was confirmed that both antibodies have neurotoxicity neutralizing activity. Both antibodies also confirmed Aβ amyloid fibril formation inhibitory activity in thioflavin T assay and electron microscopy. The ability of both antibodies to capture Aβ oligomers in AD brain was confirmed by the presence of SDS-stable 4-, 5-, 8- and 12-mers by immunoprecipitation using 1A9 and 2C3. Furthermore, for the purpose of verifying in vivo neurotoxicity in the human brain, the number of polymers recognized by both antibodies was verified mainly in the human entorhinal cortex of Braak NFT stages I to III. In particular, as a result of focusing on 12-mer, which was reported to have neurotoxicity in animal experiments, it was confirmed that the accumulation preceded the appearance of cognitive dysfunction and increased as the Braak NFT stage progressed. This result shows for the first time that the 12-mer specifically recognized by both antibodies is a conformational assembly responsible for in vivo neurotoxicity in the human brain. The inventors have also found that oligomeric conformations recognized by both antibodies are present in CSF (cerebrospinal fluid) and are increased in AD patients. We used 1A9 or 2C3 as a passive immunotherapy by intravenous injection as well as other neurological diseases, and subchronic passive immunization resulted in Tg2576 mice having memory impairment, senile plaque lesions, synaptic dysfunction and Aβ It was confirmed that it was protected from accumulation and had no harmful side effects. Our results show that monoclonal 1A9 or 2C3 is effective as a therapeutic antibody for preventing the Alzheimer-like phenotype in Tg2576 mice, especially in normal peripheral vein administration without considering brain entry. This is the first time that it is a promising candidate that can be expected.

  The present inventors have also confirmed that senile plaque amyloid and swollen neurite formation are suppressed by passive immunotherapy with 1A9 and 2C3 antibodies. Furthermore, it has been found that some of the 1A9 and 2C3 antibodies administered into the blood migrate into the brain.

  As described above, the present inventors herein used a treatment for diagnosing or preventing Alzheimer's disease when monoclonal 1A9 or 2C3, which is an antibody that specifically binds to an Aβ oligomer, meets all diagnostic / therapeutic antibody setting criteria. It is presented as a promising candidate for antibodies.

  In addition, the present inventors succeeded in obtaining antibodies 5A5, 5A9, 4F7, 4H5, 6E4 and 6H4 that specifically recognize the Aβ oligomer without recognizing the Aβ monomer, similarly to the 1A9 and 2C3 antibodies. Six types of antibodies were found to have Aβ-induced neurotoxicity neutralizing activity and Aβ amyloid fibril formation inhibitory activity.

  Thus, the 5A5, 5A9, 4F7, 4H5, 6E4 and 6H4 antibodies described above are promising candidates for therapeutic antibodies for diagnosing and preventing Alzheimer's disease.

That is, the present invention
[1] An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 1 and an L chain having the amino acid sequence of SEQ ID NO: 3 binds,
[2] An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 21 and an L chain having the amino acid sequence of SEQ ID NO: 23 binds,
[3] An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 41 and an L chain having the amino acid sequence of SEQ ID NO: 43 binds,
[4] An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 61 and an L chain having the amino acid sequence of SEQ ID NO: 63 binds;
[5] An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 81 and an L chain having the amino acid sequence of SEQ ID NO: 83 binds;
[6] An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 101 and an L chain having the amino acid sequence of SEQ ID NO: 103 binds,
[7] The antibody according to any one of (1) to (38) below,
(1) An antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 9 as CDR1, the amino acid sequence of SEQ ID NO: 11 as CDR2, and the amino acid sequence of SEQ ID NO: 13 as CDR3 (2) CDR1 As an antibody comprising an L chain having the amino acid sequence of SEQ ID NO: 15 as SEQ ID NO: 17 as CDR2, the amino acid sequence of SEQ ID NO: 17 as CDR3, and the amino acid sequence of SEQ ID NO: 19 as CDR3 (4) an antibody comprising the H chain having the amino acid sequence described in SEQ ID NO: 5 as VH (5) an amino acid described in SEQ ID NO: 7 as VL An antibody comprising an L chain having a sequence (6) An H chain according to (4), and an antibody comprising an L chain according to (5) (7) The amino acid sequence of SEQ ID NO: 29 as CDR1 and the sequence as CDR2 Number: 31 amino acid sequence And an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 33 as CDR3 (8) the amino acid sequence set forth in SEQ ID NO: 35 as CDR1, the amino acid sequence set forth in SEQ ID NO: 37 as CDR2, and the CDR3 The antibody (9) containing the L chain having the amino acid sequence shown in SEQ ID NO: 39 (9) The H chain described in (7), and the antibody (10) containing the L chain described in (8) (10) as VH in SEQ ID NO: 25 An antibody (11) comprising an H chain having the amino acid sequence described in (11) an VL comprising the L chain having the amino acid sequence described in SEQ ID NO: 27 as VL, and the H chain described in (10), and (11) (13) An amino acid sequence described in SEQ ID NO: 49 as CDR1, an amino acid sequence described in SEQ ID NO: 51 as CDR2, and an H chain having the amino acid sequence described in SEQ ID NO: 53 as CDR3 Including antibodies ( 4) An antibody (15) comprising an L chain having the amino acid sequence of SEQ ID NO: 55 as CDR1, the amino acid sequence of SEQ ID NO: 57 as CDR2, and the amino acid sequence of SEQ ID NO: 59 as CDR3 (13) An antibody comprising the H chain described in (14) and the L chain described in (14) (16) An antibody comprising the H chain having the amino acid sequence described in SEQ ID NO: 45 as VH (17) as SEQ ID NO: 47 as VL An antibody comprising the L chain having the amino acid sequence described (18), the H chain according to (16), and the antibody comprising the L chain according to (17) (19) the amino acid sequence represented by SEQ ID NO: 69 as CDR1; An antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 71 as CDR2 and the amino acid sequence set forth in SEQ ID NO: 73 as CDR3 (20) The amino acid sequence set forth in SEQ ID NO: 75 as CDR1 and SEQ ID NO: CDR2 An antibody comprising an L chain having the amino acid sequence set forth in SEQ ID NO: 79 as CDR3 and the H chain set forth in (19) and the L chain set forth in (20) Antibody (22) Antibody (23) comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 65 as VH (23) Antibody (24) (22) comprising an L chain having the amino acid sequence set forth in SEQ ID NO: 67 as VL An antibody comprising the H chain described above and the L chain described in (23) (25) amino acid sequence set forth in SEQ ID NO: 89 as CDR1, amino acid sequence set forth in SEQ ID NO: 91 as CDR2, and SEQ ID NO: as CDR3 An antibody comprising an H chain having the amino acid sequence of 93 (26) The amino acid sequence of SEQ ID NO: 95 as CDR1, the amino acid sequence of SEQ ID NO: 97 as CDR2, and the SEQ ID NO: 99 of CDR3 A An antibody comprising an L chain having a mino acid sequence (27) having the amino acid sequence set forth in SEQ ID NO: 85 as an H chain described in (25) and an antibody (28) VH comprising the L chain described in (26) Antibody comprising an L chain having the amino acid sequence set forth in SEQ ID NO: 87 as an antibody comprising an H chain (29) VL (30) An antibody comprising the H chain according to (28) and the L chain according to (29) (31) An antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 109 as CDR1, the amino acid sequence of SEQ ID NO: 111 as CDR2, and the amino acid sequence of SEQ ID NO: 113 as CDR3 (32) CDR1 As an antibody comprising an L chain having the amino acid sequence described in SEQ ID NO: 115, the amino acid sequence described in SEQ ID NO: 117 as CDR2, and the amino acid sequence described in SEQ ID NO: 119 as CDR3 (33) (31) of An antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 105 as an antibody (34) VH comprising the H chain and the L chain set forth in (32) (35) The amino acid sequence set forth in SEQ ID NO: 107 as a VL In the antibody according to any one of (37) (1) to (36), comprising an H chain according to (36) (34) and an L chain according to (35). Or an antibody in which a plurality of amino acids are substituted, deleted, added and / or inserted, and has an activity equivalent to that of the antibody according to any one of (1) to (36) (38) (1) An antibody that binds to an epitope to which the antibody according to any one of (36) binds [8] The antibody according to [7], wherein the antibody is a chimeric antibody or a humanized antibody,
[9] A composition comprising the antibody according to any one of [1] to [8] and a pharmaceutically acceptable carrier,
[10] An anticognitive dysfunction agent comprising the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[11] A therapeutic agent for Alzheimer's disease comprising the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[12] Alzheimer's progression inhibitor containing the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[13] A senile plaque formation inhibitor comprising the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[14] Aβ accumulation inhibitor containing the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[15] An anti-neurotoxic agent comprising the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[16] An Aβ amyloid fibril formation inhibitor comprising the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[17] An antisynaptic toxicant comprising the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[18] A method for measuring Aβ oligomer, comprising a step of detecting Aβ oligomer contained in a sample collected from a subject using the antibody according to any one of [1] to [8],
[19] Diagnosis as to whether or not a subject is a candidate for Alzheimer's disease, wherein Aβ oligomers in a sample collected from the subject are detected using the antibody according to any one of [1] to [8] how to,
[20] A method for diagnosing whether or not a subject is a candidate for Alzheimer's disease comprising the following steps, wherein the subject is Alzheimer's disease when the amount described in step (b) is higher than that of a healthy subject A method that is determined to be a candidate,
(A) a step of contacting a sample collected from a subject and the antibody according to any one of [1] to [8] (b) a step of measuring the amount of Aβ oligomer in the sample [21] the following steps The subject is determined to be an Alzheimer's disease candidate when the ratio described in step (b) is higher than that of a healthy subject. How to
(A) a step of contacting a sample collected from a subject, the antibody according to any one of [1] to [8], and an antibody that binds to Aβ monomer; (b) a ratio of Aβ oligomer to Aβ monomer in the sample; [22] The method according to any one of [18] to [21], wherein the sample is blood or cerebrospinal fluid,
[23] A drug for use in the method according to any one of [18] to [21],
[24] A kit for use in the method according to any one of [18] to [21],
Is to provide.

Furthermore, the present invention provides
[25] Either or both of prevention and treatment of cognitive dysfunction, comprising the step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient Way for the
[26] Either or both of the prevention and treatment of Alzheimer's disease comprising the step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient Way for,
[27] A method for suppressing progression of Alzheimer's disease, comprising a step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[28] A method for suppressing senile plaque formation, comprising a step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[29] A method for suppressing Aβ accumulation, comprising a step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[30] A method for neutralizing neurotoxicity, comprising a step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[31] A method for inhibiting the formation of Aβ amyloid fibrils comprising the step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[32] A method for neutralizing synaptic toxicity comprising the step of administering the antibody according to any one of [1] to [8] or the composition according to [9] as an active ingredient,
[33] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the manufacture of an antidementia dysfunction agent,
[34] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the manufacture of a therapeutic agent for Alzheimer's disease,
[35] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the manufacture of an Alzheimer's disease progression inhibitor,
[36] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the manufacture of a senile plaque formation inhibitor;
[37] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the manufacture of an Aβ accumulation inhibitor,
[38] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the production of a neurotoxic activity neutralizing (suppressing) agent,
[39] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the manufacture of an Aβ amyloid fibril formation inhibitor,
[40] Use of the antibody according to any one of [1] to [8] or the composition according to [9] in the production of a synaptic toxicity activity neutralizing (suppressing) agent,
[41] The antibody according to any one of [1] to [8] or the composition according to [9] for use in either or both of prevention and treatment of cognitive dysfunction,
[42] The antibody according to any one of [1] to [8] or the composition according to [9] for use in either or both of prevention and treatment of Alzheimer's disease,
[43] The antibody according to any one of [1] to [8] or the composition according to [9] for use in suppressing progression of Alzheimer's disease,
[44] The antibody according to any one of [1] to [8] or the composition according to [9] for use in suppressing senile plaque formation;
[45] The antibody according to any one of [1] to [8] or the composition according to [9] for use in suppressing Aβ accumulation,
[46] The antibody according to any one of [1] to [8] or the composition according to [9] for use in neutralizing (suppressing) neurotoxic activity;
[47] The antibody according to any one of [1] to [8] or the composition according to [9] for use in inhibiting Aβ amyloid fibril formation,
[48] The antibody according to any one of [1] to [8] or the composition according to [9] for use in neutralizing (suppressing) synaptic toxic activity,
I will provide a.

  The antibody provided by the present invention is expected to make a great contribution to the establishment of Alzheimer's disease-causing responsible molecule-selective prevention / treatment method and early diagnosis marker. As for the antibody therapy that should target the pathological condition in the brain, it is considered that the peripheral venous therapy is sufficient without considering its intracerebral transfer, and as a result, antibody therapy for Alzheimer's disease is expected to accelerate at a stretch. .

2 is a photograph and graph showing the results of preparation and characterization of oligomer-specific antibodies. A, electrophoresis of immunogen. Aβ1-42 tetramer (black arrow) without contamination of Aβ1-42 monomer (open arrow) was isolated by SDS-PAGE. Lane 1, Aβ1-42 dissolved in 10 mM phosphate buffer; Lane 2, Aβ1-42 dissolved in distilled deionized water. B, Aβ amyloid that is buffer-insoluble and extractable with formic acid from the brain of Alzheimer's disease patients is immunoprecipitated using the supernatant of the positive hybridoma cell culture, and the immune complex is selectively expressed with protein-G agarose (Amersham). separated. Of the 9 clones tested, lane 2 (asterisk) is 1A9 and lane 6 is 2C3 (double asterisk). C, Elution profile at SEC of conditioned media. Of the 24 fractions collected by SEC, fractions 8, 13 and 16 were subjected to 1A9 immunoprecipitation. Aβ immunoreactivity was detected by 4G8. Black arrows indicate trimmers and white ones indicate dimers. * Indicates the light chain of anti-mouse IgG. It is the photograph and graph which show the antitoxic activity of 1A9 or 2C3. Representative images of NGF-treated PC12 (PC12N) cells exposed to seed-free Aβ1-42 for 48 hours at 37 ° C. in the absence or presence of A-F, antibody (left half of each panel). Representative calcein AM / PI staining (right half of each panel) with live cells showing green staining and dead cells showing red staining. G, cell viability exposed to seed-free Aβ1-42 (25 μM): non-specific IgG2b (■ black square); 4G8 (Δ white triangle); 1A9 (□ white square); 2C3 (● black circle). 1 is a photograph and graph showing the size and morphological characteristics of a toxic Aβ polymer targeted by 1A9 or 2C3. A, Aβ1-42 540,000 × g supernatant (25 μM) was subjected to a continuous molecular sieving step using membrane ultrafiltration membranes with molecular weight cut-off values of 3, 10, 30 and 100 kDa (Microcon). The resulting four fractionated filtrates were named as follows: fraction 1 (<3 kDa), fraction 2 (3-10 kDa), fraction 3 (10-30 kDa) and fraction 4 ( 30-100 kDa) as well as the final retentate fraction 5 (> 100 kDa). The presence of Aβ1-42 in all the above fractions was examined sequentially by 4G8 immunoblot. B, Representative image of NGF treated PC12 (PC12N) cells treated with 5 fractions for 48 hours at 37 ° C. The toxicity of each fraction was evaluated as described in FIG. C, Viability of cells treated with 540,000 × g supernatant of Aβ1-42 and 5 fractions (fractions 1-5). Similar results were obtained by two independent experiments. Values are expressed as a percentage of the control (mean ± SD). D, dot blot analysis of 5 fractions (fractions 1-5). The blot was reacted with A11, 1A9, 2C3 and 4G8. E, AFM image of 5 different fractions. In the most toxic Fr. 5 (Fr. 5), in addition to particulate molecules, a ring-like or beaded form was also observed. 2 is a photograph and a graph showing Aβ amyloid fibril formation inhibitory activity of 1A9 or 2C3. A, Aβ1-42 amyloid fibril formation at various concentrations (10, 25, 50 μM) was monitored in a ThT assay at 37 ° C. for up to 72 hours: 10 μM (□ white squares); 25 μM (◆ Black diamonds) ); 50 μM (○ white circle). B, co-antibody dose-dependent inhibition of Aβ1-42 amyloid fibril formation was observed in 2C3 (○), but 1A9 (□ white square), 4G8 (▲ black triangle) and non-specific IgG (■ black square) None of these antibodies inhibited fibrogenic polymerization of seed-free Aβ1-42 (ThT-negative supernatant with 540,000 × g). Co-antibody dose-dependent inhibition of fibrogenic polymerization of C and Aβ1-42 was observed with 2C3 (◯), and almost complete inhibition was observed with 1A9 (□ white squares) at 3 μM. D. None of the test antibodies added after incubating in advance for 24 hours to form amyloid fibril formation of Aβ1-42 failed to dissolve or depolymerize amyloid fibrils of Aβ1-42. EM images of Aβ1-42 in the absence (panel E) and presence (panel F, panel G, respectively) of EG, 2C3 and 1A9. 2 is a photograph and graph relating to a toxicity-related Aβ1-42 oligomer. A, dot blot assay (upper half of panel A): Aβ1-42 monomer (25 μM) incubated at 37 ° C. for the specified time (0-72 hours) was immobilized on a nitrocellulose membrane and A11, 1A9, 2C3 Alternatively, they were subjected to a dot blot assay using 4G8 to verify the appearance of immunoreactive positive structures of each antibody. Immunoactivity intensity analysis (lower half of panel A): The results of the dot blot assay were semi-quantitatively analyzed by Multi Gauge v3.0 software (Fuji Film, Tokyo). In order to compare the relationship between oligomer formation and amyloid fibril formation, ThT fluorescence values (Y axis on the right) were overlaid on the same time axis. B, Aβ1-42 polymerization state at 0, 2, 4, 24 hours incubation at 37 ° C and Aβ1-42 polymerization change at 48 hours incubation. Aβ1-42 polymerization state was detected by 4G8 immunoblot. C, Toxic activity of the various Aβ1-42 polymers described above. Neuronal viability was determined by LIVE / DEAD assay as described in FIG. D, 1A9 or 2C3 anti-neurotoxic activity with various Aβ polymers (Aβ1-42 polymer [0h, 2h] formed at 37 ° C, 0, 2 hours; ThT recovered after ultracentrifugation at 540,000 × g for 2 hours A positive supernatant [2h sup]) was used for verification. Representative images of PC12N cells exposed to various Aβ1-42 polymers in the absence or presence of antibodies are shown in the left half of panel D (a: 0h; b: 2h; c: 2h sup; d: 2h sup + IgG2b; e: 2h sup + 1A9; f: 2h sup + 2C3). Cell viability exposed to various Aβ1-42 polymers in the absence or presence of antibody was expressed as a percentage of the control (mean ± SD) and is shown in the right half of panel D. Aβ1-42 polymer (2h) reduced neurotoxicity compared to Aβ1-42 polymer (0h), but 2hsup recovered neurotoxicity to the same extent as Aβ1-42 polymer (0h). Nonspecific IgG2b failed to block the induction of neurotoxicity of Aβ1-42 polymer [2h sup]. Monoclonal 1A9 completely inhibited 2hsup-induced neurotoxicity, but 2C3's ability to inhibit toxicity was somewhat inferior. On the other hand, in both monoclonal experiments, the antitoxic activity of both mAbs was observed at a molar ratio of mAb to Aβ of less than 1: 25-50, which is structurally different oligomeric 1A9 or 2C3 polymer Is present at relatively low concentrations. 2 is a photograph and graph showing that soluble 1A9 or 2C3 oligomers are present in the human brain. Antibodies against Aβ oligomers can detect senile plaques and vascular amyloid in the AD brain only after protease-K pretreatment. A, 1A9 staining; B, 2C3 staining; C, A11 staining. D, 4G8 immunoblot of 1A9 or 2C3 immunoprecipitated Aβ in buffer soluble AD brains (lanes 1-2, 4-5) and healthy control brains (lanes 3, 6). Representative results for 1A9 in the left half of the panel and 2C3 in the right half of the panel are shown. E and F, obtained from 50 autopsy cases of healthy elderly population (Braak NFT stage I-II, n = 35; Braak NFT stage III-IV, n = 13, Braak NFT stage> IV, AD case, n = 2) Analysis of soluble 1A9 immunoreactive 12-mer (panel E) and soluble 2C3 immunoreactive 12-mer (panel F) in human entorhinal cortex (corrected with actin). Figure 2 is a graph showing that soluble 1A9 or 2C3 oligomers are present in human CSF. Panels A and B show pooled whole CSF (AD = 10, NC = 10), and Panels C and D show pooled lipoprotein-depleted CSF (AD = 10, NC = 10) was subjected to size exclusion chromatography (SEC). In panels A and B, the collected fractions were analyzed for Aβ40 monomer and Aβ42 monomer distribution using BNT77-BA27 and BNT77-BC05 ELISAs. Panels C and D show the presence of Aβ40 oligomers and A42 oligomers captured with the 1A9 / 2C3 mixed antibody. FIG. 7 is a continuation diagram of FIG. The amount of oligomeric 1A9 polymer (1A9-BC05, 1A9-BA27 ELISAs) or 2C3 polymer (2C3-BC05, 2C3-BA27 ELISAs) was measured in 12 AD cases (○ white circle) and 13 NC cases (● black circle). (Panels E and G). The ratio of oligomer to monomer is shown in Figures F (1A9) and H (2C3), respectively. It is a graph which shows that the memory disorder onset of Tg2576 mouse | mouth can be prevented by passive immunization. 13 months old Tg2576 mice were divided into the following 3 groups, and learning and behavioral tests were performed: PBS administration group, n = 10; 1A9 administration group, n = 13; 2C3 administration group, n = 11. All measured values are shown as mean ± SE. (A) Y maze test. Spontaneous change behavior during an 8-minute session in the Y maze task was measured in each group. Results with one-way ANOVA are as follows: F (1, 52) = 3.09, p <0.05, * p <0.05 compared to Tg2576 mice administered with PBS. (B) New object recognition test. Retention sessions were conducted 24 hours after training. Exploratory preference during a 10 minute session in the novel object recognition test was measured in each group. Results with two-way ANOVA were as follows: training / retention, F (1,64) = 31.53, p <0.01; animal group, F (2,64) -7.49, p <0.01; depending on animal group Repeated training / retention, F (2,64) = 10.12, p <0.01; ** p <0.01 compared to the corresponding untrained mouse. ## p <0.01 compared to Tg2576 mice administered with PBS. (C) The length of the swimming path during the 60-second session in the water maze test was measured for each group. Results with two-way ANOVA were as follows: trial, F (9,320) = 2.46, p <0.01; animal group, F (2,320) = 12.59, p <0.01; repeated trial with animal group, F ( 18,320) = 1.78, p <0.05; p <0.05, ** p <0.01 compared to Tg2576 mice administered with PBS. Conditioned fear learning test: Context-dependent (D) and cue-dependent freezing time (E) were measured. Results from two-way ANOVA were as follows: context-dependent test, F (2,32) = 5.94, p <0.01, cue-dependent test, F (2,32) = 7.33, p <0.01; * PBS P <0.05 compared to Tg2576 mice treated with **, and ** is also p <0.01. It is the photograph and graph which show that Tg2576 brain A (beta) accumulation prevention is possible by passive immunotherapy. Three groups of 13-month-old Tg2576 mice (PBS-administered group, n = 10; 1A9-administered group, n = 13; 2C3-administered group, n = 11) were extracted in three stages, and buffer soluble (saline -soluble) fraction, SDS-soluble fraction, and formic acid extract (FA-extractable) fraction were prepared. When each fraction was measured by Aβ-specific ELISAs (WAKO kit: BNT77-BA27 for Aβx-40; BNT77-BC05 for Aβx-42), SDS40 / 42 and FA40 accumulation was significantly suppressed only in the 1A9 treatment group. It became clear that. In SDS-soluble cerebral cortex fractions, the accumulation inhibitory effect of A11 positive oligomer (4-mer) was confirmed in both antibody treatment groups. It is the photograph and graph which are shown about the A (beta) oligomer in the plasma and brain of Tg2576. As a result of A, B, and ELISA analyses, no significant changes were observed in the amounts of Aβx-40 and Aβx-42 in plasma even when the PBS administration group and the immunotherapy group were compared. Similarly, the ratio of C and Aβ40 / Aβ42 did not change in the three groups tested. D, analysis by dot blot with pooled brain homogenates showed no change in the amount of saline soluble A11 positive oligomers in the three groups tested: hippocampus (left panel) and cerebral cortex (right panel). PBS administration group (n = 10), 1A9 administration group (n = 13), 2C3 administration group (n = 11). E. According to immunoblot analysis using anti-oligomer antibody A11, the immune reaction of Aβ tetramer in the SDS-extracted cerebral cortex fraction (right panel) was decreased in the 1A9 or 2C3 administration group compared to the PBS administration group. On the other hand, such a phenomenon was not confirmed in the hippocampus (left panel). F, blood (albumin-removed plasma, upper panel F; albumin / lipoprotein-removed plasma, lower panel F) within each group were pooled and subjected to A11 dot blot analysis. As a result, 1A9 and 2C3 Increased A11 immunoreactivity was observed in the administration group (Panel F). In addition, 2C3 oligomers were present in a higher proportion in the lipoprotein-binding form than 1A9 oligomers (lower panel F). Furthermore, the A11 immunoblot was positive at about 200 kDa, and its immunoreactivity was clearly increased in the 1A9 and 2C3 administration groups compared to the PBS administration group (Panel G). This result can be regarded as a result in which a selective therapeutic effect is expressed only in the target Aβ oligomer molecule without affecting physiological molecules in the antibody administration group. Shows that passive immunization suppressed senile plaque amyloid (A: Aβ-specific antibody staining, B: thioflavin-S positive analysis) and swollen neurite formation in the brain of Tg2576 mice (C: synaptophysin positive analysis) It is a figure and a photograph. It is a photograph showing the suppression of synaptic degeneration by passive immunization treatment with 1A9 or 2C3. Immunostaining with synaptophysin (left panel) or drebrin (right panel) in pre- or post-synaptic punctate peripheral cells. Upper figure: PBS administration, middle figure: 1A9 administration, lower figure: 2C3 administration. It is a photograph shown about the brain transfer of the antibody by passive immunization. Photo of distribution of antibody administered in Tg2576 mouse brain. Stained image with anti-Aβ antibody (left panel), IgG stained image (center panel). 1A9 administration (A), 2C3 administration (B), PBS administration (C). It is the photograph which confirmed that the monoclonal antibodies 5A5, 5A9, 4F7, 4H5, 6E4 and 6H4 were specific to Aβ oligomer (3-96 hr) and did not recognize Aβ monomer (0 hr) by dot blot analysis. It is the graph which showed the Abeta oligomer selective binding ability of six types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4). The vertical axis indicates the absorbance at a wavelength of 450 nm, and the horizontal axis indicates the concentration of Aβ oligomer or Aβ monomer used as an inhibitor. The broken line in each graph shows the antibody binding activity using Aβ oligomer as an inhibitor, and the solid line shows the antibody binding activity when Aβ monomer is used as an inhibitor. It is a graph which shows the A (beta) induced neurotoxicity neutralizing activity of 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4). The horizontal axis shows the amount of antibody added, and the vertical axis shows the cytotoxicity relative to the condition in which no antibody was added (see the calculation formula in the figure). Control IgG (3F1) is an antibody that does not bind to Aβ42 and was used as a comparison target. It is a graph which shows the Abeta amyloid fibril formation inhibitory activity of six types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4). The antibody was added to the Aβ1-42 (12.5 μM) solution at three concentrations and incubated at 37 ° C. for 24 hours, and then the amount of Aβ amyloid fibril formation was measured by ThT fluorescence intensity measurement. The abscissa represents the amount of antibody added, and the ordinate represents the numerical value relative to the amount of amyloid fibril formation at the time of antibody addition based on the amount of antibody-free Aβ amyloid fibril formation.

[Embodiment of the Invention]
The present invention is described in further detail below.

  As described above, the present inventors have succeeded in obtaining an antibody that specifically binds to an Aβ oligomer and does not bind to an Aβ monomer. That is, the present invention provides an antibody that binds to an Aβ oligomer and does not bind to an Aβ monomer. Such antibodies are preferably isolated antibodies or purified antibodies.

  The terms “isolated” and “purified” as used in a substance (such as an antibody) of the present invention substantially refer to at least one substance that may be contained in a natural source. Is not included. Thus, an “isolated antibody” or “purified antibody” refers to cellular material such as hydrocarbons, lipids, or other contaminating proteins from the cell or tissue source from which the antibody (protein) is derived. Refers to an antibody that is substantially free or, if chemically synthesized, substantially free of chemical precursors or other chemicals. In preferred embodiments, the antibodies of the invention are isolated or purified.

  An “antibody” is a glycoprotein having the same structural characteristics. An antibody exhibits binding specificity for a particular antigen. As used herein, “antigen” refers to a protein that has the ability to bind to a corresponding antibody and induces an antigen-antibody reaction in vivo.

  Aβ protein, the main component of amyloid, is a 40-42 amino acid peptide that is known to be produced by the action of proteases from a precursor protein called amyloid precursor protein (APP). Yes. Apart from amyloid fibrils collected in ultracentrifugation fractions, amyloid molecules produced from APP include non-fibrous polymers that are oligomers in addition to soluble monomers. The “Aβ oligomer” in the present invention refers to a non-fibrous polymer. The “Aβ oligomer” in the present invention includes, for example, an Aβ40 (Aβ1-40) oligomer and an Aβ42 (Aβ1-42) oligomer. For example, the “Aβ42 oligomer” in the present invention is a molecule having a molecular weight of 45 to 160 kDa in SDS-PAGE and a molecular weight of 22.5 to 1,035 kDa in Blue Native-PAGE. Molecular sieves are mainly recovered in a retentate> 100 kDa, and form a mixture of granular, beaded, and ring-shaped molecules 1.5-3.1 nm in height with an atomic force microscope. In the gel filtration method, it was eluted in void volume fraction 8 with a molecular weight> 680 kDa and fraction 15 at the molecular weight 17-44 kDa boundary.

  The antibody used in the present invention may be any antibody that binds to the Aβ oligomer and does not bind to the Aβ monomer, and its origin and shape are not limited.

  The “antibody” in the present invention includes both monoclonal antibodies and polyclonal antibodies. In addition, the antibodies of the present invention include non-human animal antibodies, humanized antibodies, chimerized antibodies, human antibodies, low molecular weight antibodies described below, antibodies with modified amino acid sequences, other molecules (for example, polyethylene glycol, etc. Any antibody such as a modified antibody to which a polymer or the like is bound, or an antibody with a modified sugar chain is included.

  As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical except for possible natural mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and correspond to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies to different antigenic determinants (epitopes), each monoclonal antibody corresponds to a single determinant on the antigen. .

  In addition to the specificities described above, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cultures that are not contaminated with other immunoglobulins. Thus, “monoclonal” indicates the character of the antibody as obtained from a substantially homogeneous antibody population, and should not be construed as requiring production of the antibody by any particular method.

  Monoclonal antibodies can be basically produced using known techniques. For example, it may be made by the hybridoma method first described by Kohler and Milstein (Nature 256: 495-7, 1975) or by the recombinant DNA method (Cabilly et al., Proc Natl Acad Sci USA 81: 3273-7 1984), but is not limited by these methods. For example, when the hybridoma method is used, an Aβ oligomer (for example, Aβ tetramer as described in the Examples) is used as a sensitizing antigen, and this is immunized according to a normal immunization method. It can be prepared by fusing with a known parent cell by a fusion method and screening monoclonal antibody-producing cells by a conventional screening method.

  The monoclonal antibody in the present invention can be prepared as follows. First, synthetic Aβ1-42 (Peptide Laboratories, Osaka) was dissolved in distilled deionized water or 10 mM phosphate buffer as an antigen, incubated at 37 ° C for 18 hours, and then 4-12% SDS-PAGE. The peptide is isolated and visualized by CBB staining, then only the Aβ1-42 tetramer without Aβ1-42 monomer contamination is cut out, or the synthesized Aβ1-40 peptide has 6-Carboxytetramethylrhodamine (6-TAMRA) at the N-terminus Modified Aβ1-40 (SIGMA) chemically bound using conventional methods and synthetic Aβ1-40 (Peptide Laboratories, Osaka) 5: 100, 10: 100, 20: 100, 30: 100, 40: 100, 50: 100, 60: 100, 70: 100, or 80: 100, preferably 90: 100, more preferably 100: 100, 3 hours, preferably 6 hours, more preferably 20 hours polymerization reaction To prepare a preparation (Aβ1-40 oligomer) rich in oligomers. Next, Balb-c mice were immunized with 2.5 μg of Aβ1-42 tetramer or Aβ1-40 oligomer emulsified with complete Freund's adjuvant, followed by 6 additional additions. Immunize. Hybridomas are prepared by fusion with Sp2 / O-Ag14 cells using polyethylene glycol 1500 using inguinal lymph nodes.

  The animal to be immunized with the sensitizing antigen is not particularly limited, and may be selected in consideration of compatibility with the parent cell used for cell fusion. Generally, rodents, rabbit eyes, or primates Kind is used. Rodents include, for example, mice, rats, and hamsters. Rabbit eyes include, for example, rabbits. Primates include, for example, nasal monkey (old world) monkeys such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), baboons, and chimpanzees.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, the sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously.

  Examples of the other parent cell that fuses with the immune cells include Sp2 / O-Ag14 cells described in Examples below, but various other known cell lines can also be used.

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein (Kohler G. and Milstein C., Methods Enzymol. (1981) 73, 3-46). It can carry out according to etc.

  The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Cultivation with the above HAT culture solution is usually continued for a period of several days to several weeks for a time sufficient for cells other than the target hybridoma (non-fused cells) to die. Subsequently, a normal limiting dilution method is performed, and screening and single cloning of a hybridoma that produces the target antibody are performed.

  Thereafter, the obtained hybridoma is transplanted into the abdominal cavity of a mouse, and ascites containing the target monoclonal antibody is extracted. Conventional, including selected combinations of column chromatography including, but not limited to, affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. Antibodies can be purified from ascites by protein separation and / or purification methods (Antibodies: A Laboratory Manual, Harlow and David, Lane (edit.), Cold Spring Harbor Laboratory, 1988).

  Protein A and protein G columns can be used as affinity columns. Exemplary protein A columns used include Hyper D, POROS, and Sepharose F.F. (Pharmacia).

  Chromatography (excluding affinity chromatography) includes ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography ("Strategies for Protein Purification and Characterization: A Laboratory Course Manual "Daniel R Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be performed according to liquid phase chromatography procedures such as HPLC and FPLC.

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In addition, any mammal can be immunized using an immunogen for antibody production. However, when preparing a monoclonal antibody by producing a hybridoma, it is preferable to consider compatibility with the parent cell used in cell fusion for hybridoma production.

  In general, rodents, rabbit eyes, or primates are used for such immunization. Rodents include, for example, mice, rats, and hamsters. Rabbit eyes include, for example, rabbits. Primates include, for example, nasal monkey (old world) monkeys such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), baboons, and chimpanzees.

  The use of transgenic animals containing a repertoire of human antibody genes is known in the art (Ishida I, et al., Cloning and Stem Cells 4: 91-102, 2002). As with other animals, to obtain human monoclonal antibodies, transgenic animals are immunized, antibody-producing cells are then collected from the animals, fused with myeloma cells to obtain hybridomas, and these hybridomas. Anti-protein human antibodies can be prepared from (see International Publication Nos. 92-03918, 94-02602, 94-25585, 96-33735, and 96-34096).

  Alternatively, lymphocytes immortalized with oncogenes can be used for monoclonal antibody production. For example, human lymphocytes infected with EB virus or the like can be immunized in vitro with an immunogen. Next, immunized lymphocytes are fused with human-derived myeloma cells (such as U266) that can divide indefinitely, thus obtaining a hybridoma that produces the desired human antibody (Japanese Patent Application Publication) Publication (JP-A) Sho 63-17688).

  Once a monoclonal antibody can be obtained via any of the methods described above, it can also be prepared using genetic engineering methods (eg, Borrebaeck CAK and Larrick JW, Therapeutic Monoclonal Antibodies, MacMillan Publishers, UK, 1990). For example, recombinant antibodies can be obtained by cloning DNA encoding the antibody of interest from antigen producing cells such as antibody-producing hybridomas or immunized lymphocytes, and then inserting the cloned DNA into an appropriate vector. Can be prepared by transforming the vector into a suitable host cell. Such recombinant antibodies are also encompassed by the present invention.

Examples of the monoclonal antibody in the present invention include 1A9 monoclonal antibody, 2C3 monoclonal antibody, 5A5 monoclonal antibody, 5A9 monoclonal antibody, 4F7 monoclonal antibody, 4H5 monoclonal antibody, 6E4 monoclonal antibody, and 6H4 monoclonal antibody. Preferably, the antibody is any of the following (i) to (vi).
(I) an antibody comprising an H chain (heavy chain) having the amino acid sequence set forth in SEQ ID NO: 1 and an L chain (light chain) having the amino acid sequence set forth in SEQ ID NO: 3 (ii) in SEQ ID NO: 21 An antibody comprising the H chain (heavy chain) having the amino acid sequence described and the L chain (light chain) having the amino acid sequence set forth in SEQ ID NO: 23 (iii) the H chain having the amino acid sequence set forth in SEQ ID NO: 41 (Heavy chain) and an antibody comprising an L chain (light chain) having the amino acid sequence set forth in SEQ ID NO: 43 (iv) an H chain (heavy chain) having the amino acid sequence set forth in SEQ ID NO: 61, and SEQ ID NO: An antibody (v) comprising an L chain (light chain) having the amino acid sequence set forth in 63 (v) an H chain (heavy chain) having the amino acid sequence set forth in SEQ ID NO: 81, and the amino acid sequence set forth in SEQ ID NO: 83 Antibody (vi) comprising L chain (light chain) having SEQ ID NO: 101 An antibody comprising an H chain (heavy chain) having an amino acid sequence and an L chain (light chain) having the amino acid sequence set forth in SEQ ID NO: 103. One embodiment of the antibody of the present invention is a low molecular weight antibody. You can also. The low molecular weight antibody is not particularly limited as long as it includes an antibody fragment in which a part of the full-length antibody is deleted and has an ability to bind to an antigen. Examples of antibody fragments include Fab, Fab ′, F (ab ′) 2, and Fv. Examples of low molecular weight antibodies include Fab, Fab ', F (ab') 2, Fv, scFv (single chain Fv), Diabody, sc (Fv) 2 (single chain (Fv) 2), and the like. be able to.

  Here, in order to obtain a polyclonal antibody against the protein of the present invention, after confirming that the desired antibody level in the serum has increased, the blood of the mammal sensitized with the antigen is taken out. Serum is separated from this blood by a known method. As a polyclonal antibody, a serum containing a polyclonal antibody may be used, and if necessary, a fraction containing a polyclonal antibody may be further isolated from this serum and used. For example, by using an affinity column coupled with the protein of the present invention, a fraction that recognizes only the protein of the present invention is obtained, and this fraction is further purified using a protein A or protein G column. Immunoglobulin G or M can be prepared.

In the present invention, the antibody that binds to Aβ oligomer is an antibody that binds to Aβ oligomer to which 1A9, 2C3, 5A5, 5A9, 4F7, 4H5, 6E4, or 6H4 binds, and preferably the following (A) to (F ).
(A) An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 1 and an L chain having the amino acid sequence set forth in SEQ ID NO: 3 binds (B) SEQ ID NO: 21 An antibody that binds to an Aβ oligomer to which an antibody comprising an H chain having the amino acid sequence described in SEQ ID NO: 23 and an L chain having the amino acid sequence described in SEQ ID NO: 23 binds (C) has the amino acid sequence described in SEQ ID NO: 41 An antibody (D) that binds to an Aβ oligomer to which an antibody comprising an H chain and an L chain having the amino acid sequence of SEQ ID NO: 43 binds, and an H chain having the amino acid sequence of SEQ ID NO: 61, and SEQ ID NO: An antibody (E) that binds to an Aβ oligomer to which an antibody comprising an L chain having the amino acid sequence described in 63 binds, and an H chain having the amino acid sequence described in SEQ ID NO: 81, and SEQ ID NO: 83 An antibody that binds to an Aβ oligomer to which an antibody containing an L chain having an amino acid sequence binds (F) An H chain having the amino acid sequence set forth in SEQ ID NO: 101, and an L chain having the amino acid sequence set forth in SEQ ID NO: 103 An antibody that binds to an Aβ oligomer to which an antibody is bound The present invention also provides an Aβ oligomer to which an antibody of the present invention binds. Preferred examples of the antibody include 1A9 monoclonal antibody, 2C3 monoclonal antibody, 5A5 monoclonal antibody, 5A9 monoclonal antibody, 4F7 monoclonal antibody, 4H5 monoclonal antibody, 6E4 monoclonal antibody, and 6H4 monoclonal antibody. Such an Aβ oligomer can be used as an antigen for preparing an antibody or as a vaccine.

As a preferable aspect of the antibody in the present invention, for example, the antibody described in any of (1) to (38) below can be mentioned.
(1) An antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 9 as CDR1, the amino acid sequence of SEQ ID NO: 11 as CDR2, and the amino acid sequence of SEQ ID NO: 13 as CDR3 (2) CDR1 As an antibody comprising an L chain having the amino acid sequence of SEQ ID NO: 15 as SEQ ID NO: 17 as CDR2, the amino acid sequence of SEQ ID NO: 17 as CDR3, and the amino acid sequence of SEQ ID NO: 19 as CDR3 (4) an antibody comprising the H chain having the amino acid sequence described in SEQ ID NO: 5 as VH (5) an amino acid described in SEQ ID NO: 7 as VL An antibody comprising an L chain having a sequence (6) An H chain according to (4), and an antibody comprising an L chain according to (5) (7) The amino acid sequence of SEQ ID NO: 29 as CDR1 and the sequence as CDR2 Number: 31 amino acid sequence And an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 33 as CDR3 (8) the amino acid sequence set forth in SEQ ID NO: 35 as CDR1, the amino acid sequence set forth in SEQ ID NO: 37 as CDR2, and the CDR3 The antibody (9) containing the L chain having the amino acid sequence shown in SEQ ID NO: 39 (9) The H chain described in (7), and the antibody (10) containing the L chain described in (8) (10) as VH in SEQ ID NO: 25 An antibody (11) comprising an H chain having the amino acid sequence described in (11) an VL comprising the L chain having the amino acid sequence described in SEQ ID NO: 27 as VL, and the H chain described in (10), and (11) (13) An amino acid sequence described in SEQ ID NO: 49 as CDR1, an amino acid sequence described in SEQ ID NO: 51 as CDR2, and an H chain having the amino acid sequence described in SEQ ID NO: 53 as CDR3 Including antibodies ( 4) An antibody (15) comprising an L chain having the amino acid sequence of SEQ ID NO: 55 as CDR1, the amino acid sequence of SEQ ID NO: 57 as CDR2, and the amino acid sequence of SEQ ID NO: 59 as CDR3 (13) An antibody comprising the H chain described in (14) and the L chain described in (14) (16) An antibody comprising the H chain having the amino acid sequence described in SEQ ID NO: 45 as VH (17) as SEQ ID NO: 47 as VL An antibody comprising the L chain having the amino acid sequence described (18), the H chain according to (16), and the antibody comprising the L chain according to (17) (19) the amino acid sequence represented by SEQ ID NO: 69 as CDR1; An antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 71 as CDR2 and the amino acid sequence set forth in SEQ ID NO: 73 as CDR3 (20) The amino acid sequence set forth in SEQ ID NO: 75 as CDR1 and SEQ ID NO: CDR2 An antibody comprising an L chain having the amino acid sequence set forth in SEQ ID NO: 79 as CDR3 and the H chain set forth in (19) and the L chain set forth in (20) Antibody (22) Antibody (23) comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 65 as VH (23) Antibody (24) (22) comprising an L chain having the amino acid sequence set forth in SEQ ID NO: 67 as VL An antibody comprising the H chain described above and the L chain described in (23) (25) amino acid sequence set forth in SEQ ID NO: 89 as CDR1, amino acid sequence set forth in SEQ ID NO: 91 as CDR2, and SEQ ID NO: as CDR3 An antibody comprising an H chain having the amino acid sequence of 93 (26) The amino acid sequence of SEQ ID NO: 95 as CDR1, the amino acid sequence of SEQ ID NO: 97 as CDR2, and the SEQ ID NO: 99 of CDR3 A An antibody comprising an L chain having a mino acid sequence (27) having the amino acid sequence set forth in SEQ ID NO: 85 as an H chain described in (25) and an antibody (28) VH comprising the L chain described in (26) Antibody comprising an L chain having the amino acid sequence set forth in SEQ ID NO: 87 as an antibody comprising an H chain (29) VL (30) An antibody comprising the H chain according to (28) and the L chain according to (29) (31) An antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 109 as CDR1, the amino acid sequence of SEQ ID NO: 111 as CDR2, and the amino acid sequence of SEQ ID NO: 113 as CDR3 (32) CDR1 As an antibody comprising an L chain having the amino acid sequence described in SEQ ID NO: 115, the amino acid sequence described in SEQ ID NO: 117 as CDR2, and the amino acid sequence described in SEQ ID NO: 119 as CDR3 (33) (31) of An antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 105 as an antibody (34) VH comprising the H chain and the L chain set forth in (32) (35) The amino acid sequence set forth in SEQ ID NO: 107 as a VL In the antibody according to any one of (37) (1) to (36), comprising an H chain according to (36) (34) and an L chain according to (35). Or an antibody in which a plurality of amino acids are substituted, deleted, added and / or inserted, and has an activity equivalent to that of the antibody according to any one of (1) to (36) (38) (1) (36) an antibody that binds to an epitope to which the antibody according to any one of

  In addition, “the amino acid sequence described in SEQ ID NO: 9 as CDR1 (sequence of HA chain CDR1 of 5A5 antibody) as described in (1)” above, and the amino acid sequence described in SEQ ID NO: 11 as CDR2 (H chain CDR2 of 5A5 antibody) And the amino acid sequence described in SEQ ID NO: 5 (the VH of the 5A5 antibody) is VH in the “H chain having the amino acid sequence described in SEQ ID NO: 13 (the sequence of the H chain CDR3 of the 5A5 antibody)” as CDR3. Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 15 as CDR1 (sequence of L chain CDR1 of 5A5 antibody) described in (2) above, and the amino acid sequence described in SEQ ID NO: 17 as CDR2 (L chain CDR2 of 5A5 antibody) And the amino acid sequence having the amino acid sequence described in SEQ ID NO: 19 as the CDR3 (the L chain CDR3 sequence of the 5A5 antibody) is referred to as the amino acid sequence described in SEQ ID NO: 7 (the VL of the 5A5 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 29 as CDR1 (sequence of H chain CDR1 of 5A9 antibody) described in (7) above, and the amino acid sequence described in SEQ ID NO: 31 as CDR2 (H chain CDR2 of 5A9 antibody) The VH in the “H chain having the amino acid sequence described in SEQ ID NO: 33 (the sequence of the H chain CDR3 of the 5A9 antibody) as CDR3” is the amino acid sequence described in SEQ ID NO: 25 (the VH of the 5A9 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 35 as the CDR1 (sequence of the L chain CDR1 of the 5A9 antibody) described in (8) above, and the amino acid sequence described in SEQ ID NO: 37 as the CDR2 (the L chain CDR2 of the 5A9 antibody) And the amino acid sequence having the amino acid sequence described in SEQ ID NO: 39 (the L chain CDR3 sequence of the 5A9 antibody) as CDR3 is the amino acid sequence described in SEQ ID NO: 27 (the VL of the 5A9 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 49 as CDR1 (sequence of H chain CDR1 of 4F7 antibody) described in (13) above, and the amino acid sequence described in SEQ ID NO: 51 as CDR2 (H chain CDR2 of 4F7 antibody) VH in the “H chain having the amino acid sequence described in SEQ ID NO: 53 as the CDR3” (sequence of the 4F7 antibody H chain CDR3) is the amino acid sequence described in the SEQ ID NO: 45 (the VH of the 4F7 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 55 as CDR1 (sequence of L chain CDR1 of the 4F7 antibody) described in (14) above, and the amino acid sequence described in SEQ ID NO: 57 as CDR2 (L chain CDR2 of 4F7 antibody) And the amino acid sequence having the amino acid sequence described in SEQ ID NO: 59 (the sequence of the L chain CDR3 of the 4F7 antibody) as CDR3 is the amino acid sequence described in SEQ ID NO: 47 (the VL of the 4F7 antibody). Can be exemplified.

  In addition, as described in the above (19), “the amino acid sequence described in SEQ ID NO: 69 as CDR1 (sequence of H chain CDR1 of 4H5 antibody)”, the amino acid sequence described in SEQ ID NO: 71 as CDR2 (H chain CDR2 of 4H5 antibody) And the amino acid sequence described in SEQ ID NO: 65 (the VH of the 4H5 antibody) as the VH in the "H chain having the amino acid sequence described in SEQ ID NO: 73 as the CDR3 (the sequence of the HH CDR3 of the 4H5 antibody)" Can be exemplified.

  In addition, as described in (20) above, “the amino acid sequence described in SEQ ID NO: 75 as CDR1 (sequence of L chain CDR1 of 4H5 antibody)” and the amino acid sequence described in SEQ ID NO: 77 as CDR2 (L chain CDR2 of 4H5 antibody) And the VL in the L chain having the amino acid sequence described in SEQ ID NO: 79 as the CDR3 (the L chain CDR3 sequence of the 4H5 antibody) is the amino acid sequence described in SEQ ID NO: 67 (the VL of the 4H5 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 89 as CDR1 (sequence of H chain CDR1 of 6E4 antibody) described in (25) above, and the amino acid sequence described in SEQ ID NO: 91 as CDR2 (H chain CDR2 of 6E4 antibody) VH in the “H chain having the amino acid sequence described in SEQ ID NO: 93 as the CDR3 (H chain CDR3 sequence of the 6E4 antibody)” as the CDR3 is the amino acid sequence described in SEQ ID NO: 85 (the VH of the 6E4 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 95 as CDR1 (sequence of L chain CDR1 of 6E4 antibody) described in (26) above, and the amino acid sequence described in SEQ ID NO: 97 as CDR2 (L chain CDR2 of 6E4 antibody) And the amino acid sequence having the amino acid sequence described in SEQ ID NO: 99 (the L chain CDR3 sequence of the 6E4 antibody) as CDR3 is the amino acid sequence described in SEQ ID NO: 87 (the VL of the 6E4 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 109 as CDR1 (sequence of H chain CDR1 of 6H4 antibody) described in (31) above, and the amino acid sequence described in SEQ ID NO: 111 as CDR2 (H chain CDR2 of 6H4 antibody) VH in the “H chain having the amino acid sequence described in SEQ ID NO: 113 as the CDR3 (the H chain CDR3 sequence of the 6H4 antibody)” as CDR3 is the amino acid sequence described in SEQ ID NO: 105 (the VH of the 6H4 antibody). Can be exemplified.

  In addition, the amino acid sequence described in SEQ ID NO: 115 as CDR1 (sequence of L chain CDR1 of 6H4 antibody) described in (32) above, and the amino acid sequence described in SEQ ID NO: 117 as CDR2 (L chain CDR2 of 6H4 antibody) ) And the L chain having the amino acid sequence described in SEQ ID NO: 119 as the CDR3 (the L chain CDR3 sequence of the 6H4 antibody) is the VL in the amino acid sequence described in SEQ ID NO: 107 (the VL of the 6H4 antibody). Can be exemplified.

  The amino acid sequence of the full length H chain of the 5A5 antibody in the present invention is SEQ ID NO: 1, the base sequence is SEQ ID NO: 2, the full length L chain is SEQ ID NO: 3, the base sequence is SEQ ID NO: 4, and the H chain variable region The amino acid sequence of (VH) is SEQ ID NO: 5, the nucleotide sequence is SEQ ID NO: 6, the amino acid sequence of the L chain variable region (VL) is SEQ ID NO: 7, the nucleotide sequence is SEQ ID NO: 8, and the amino acid sequence of H chain CDR1. SEQ ID NO: 9, nucleotide sequence SEQ ID NO: 10, H chain CDR2 amino acid sequence SEQ ID NO: 11, nucleotide sequence SEQ ID NO: 12, H chain CDR3 amino acid sequence SEQ ID NO: 13, nucleotide sequence No .: 14, amino acid sequence of L chain CDR1, SEQ ID NO: 15, nucleotide sequence of SEQ ID NO: 16, amino acid sequence of L chain CDR2, SEQ ID NO: 17, nucleotide sequence of SEQ ID NO: 18, amino acid sequence of L chain CDR3 Is shown in SEQ ID NO: 19, and the nucleotide sequence is shown in SEQ ID NO: 20.

  The amino acid sequence of the full length H chain of the 5A9 antibody in the present invention is SEQ ID NO: 21, the base sequence is SEQ ID NO: 22, the full length L chain is SEQ ID NO: 23, the base sequence is SEQ ID NO: 24, and the H chain variable region. The amino acid sequence of (VH) is SEQ ID NO: 25, the base sequence is SEQ ID NO: 26, the amino acid sequence of the L chain variable region (VL) is SEQ ID NO: 27, the base sequence is SEQ ID NO: 28, and the amino acid sequence of H chain CDR1 SEQ ID NO: 29, nucleotide sequence SEQ ID NO: 30, H chain CDR2 amino acid sequence SEQ ID NO: 31, nucleotide sequence SEQ ID NO: 32, H chain CDR3 amino acid sequence SEQ ID NO: 33, nucleotide sequence No. 34, SEQ ID NO: 35 for the amino acid sequence of L chain CDR1, SEQ ID NO: 36 for the nucleotide sequence, SEQ ID NO: 37 for the amino acid sequence of L chain CDR2, SEQ ID NO: 38 for the nucleotide sequence, and amino acid sequence of L chain CDR3 Is shown in SEQ ID NO: 39, and the nucleotide sequence is shown in SEQ ID NO: 40.

  The amino acid sequence of the full length H chain of the 4F7 antibody in the present invention is SEQ ID NO: 41, the base sequence is SEQ ID NO: 42, the full length L chain is SEQ ID NO: 43, the base sequence is SEQ ID NO: 44, and the H chain variable region. The amino acid sequence of (VH) is SEQ ID NO: 45, the nucleotide sequence is SEQ ID NO: 46, the amino acid sequence of the L chain variable region (VL) is SEQ ID NO: 47, the nucleotide sequence is SEQ ID NO: 48, and the amino acid sequence of H chain CDR1. SEQ ID NO: 49, base sequence SEQ ID NO: 50, H chain CDR2 amino acid sequence SEQ ID NO: 51, base sequence SEQ ID NO: 52, H chain CDR3 amino acid sequence SEQ ID NO: 53, base sequence No .: 54, amino acid sequence of L chain CDR1, SEQ ID NO: 55, nucleotide sequence of SEQ ID NO: 56, amino acid sequence of L chain CDR2, SEQ ID NO: 57, nucleotide sequence of SEQ ID NO: 58, amino acid sequence of L chain CDR3 Is shown in SEQ ID NO: 59, and the nucleotide sequence is shown in SEQ ID NO: 60.

  The amino acid sequence of the full length H chain of the 4H5 antibody in the present invention is SEQ ID NO: 61, the base sequence is SEQ ID NO: 62, the full length L chain is SEQ ID NO: 63, the base sequence is SEQ ID NO: 64, and the H chain variable region. The amino acid sequence of (VH) is SEQ ID NO: 65, the nucleotide sequence is SEQ ID NO: 66, the amino acid sequence of the L chain variable region (VL) is SEQ ID NO: 67, the nucleotide sequence is SEQ ID NO: 68, and the amino acid sequence of H chain CDR1 SEQ ID NO: 69, nucleotide sequence SEQ ID NO: 70, H chain CDR2 amino acid sequence SEQ ID NO: 71, nucleotide sequence SEQ ID NO: 72, H chain CDR3 amino acid sequence SEQ ID NO: 73, nucleotide sequence No .: 74, amino acid sequence of L chain CDR1 is SEQ ID NO: 75, nucleotide sequence is SEQ ID NO: 76, amino acid sequence of L chain CDR2 is SEQ ID NO: 77, nucleotide sequence is SEQ ID NO: 78, amino acid sequence of L chain CDR3 Is shown in SEQ ID NO: 79, and the nucleotide sequence is shown in SEQ ID NO: 80.

  The amino acid sequence of the full length H chain of the 6E4 antibody in the present invention is SEQ ID NO: 81, the base sequence is SEQ ID NO: 82, the full length L chain is SEQ ID NO: 83, the base sequence is SEQ ID NO: 84, and the H chain variable region. The amino acid sequence of (VH) is SEQ ID NO: 85, the nucleotide sequence is SEQ ID NO: 86, the amino acid sequence of the L chain variable region (VL) is SEQ ID NO: 87, the nucleotide sequence is SEQ ID NO: 88, and the amino acid sequence of H chain CDR1. SEQ ID NO: 89, nucleotide sequence SEQ ID NO: 90, amino acid sequence of H chain CDR2 SEQ ID NO: 91, nucleotide sequence of SEQ ID NO: 92, amino acid sequence of H chain CDR3 of SEQ ID NO: 93, nucleotide sequence No. 94, amino acid sequence of L chain CDR1 SEQ ID NO: 95, nucleotide sequence of SEQ ID NO: 96, amino acid sequence of L chain CDR2 of SEQ ID NO: 97, nucleotide sequence of SEQ ID NO: 98, amino acid sequence of L chain CDR3 Is shown in SEQ ID NO: 99, and the base sequence is shown in SEQ ID NO: 100.

  The amino acid sequence of the full length H chain of the 6H4 antibody in the present invention is SEQ ID NO: 101, the base sequence is SEQ ID NO: 102, the full length L chain is SEQ ID NO: 103, the base sequence is SEQ ID NO: 104, and the H chain variable region. The amino acid sequence of (VH) is SEQ ID NO: 105, the nucleotide sequence is SEQ ID NO: 106, the amino acid sequence of the L chain variable region (VL) is SEQ ID NO: 107, the nucleotide sequence is SEQ ID NO: 108, and the amino acid sequence of H chain CDR1 SEQ ID NO: 109, nucleotide sequence SEQ ID NO: 110, H chain CDR2 amino acid sequence SEQ ID NO: 111, nucleotide sequence SEQ ID NO: 112, H chain CDR3 amino acid sequence SEQ ID NO: 113, nucleotide sequence No .: 114, amino acid sequence of L chain CDR1, SEQ ID NO: 115, nucleotide sequence of SEQ ID NO: 116, amino acid sequence of L chain CDR2, SEQ ID NO: 117, nucleotide sequence of SEQ ID NO: 118, amino acid sequence of L chain CDR3 Is shown in SEQ ID NO: 119, and the base sequence is shown in SEQ ID NO: 120.

  The antibodies described in (1) to (38) above include not only monovalent antibodies but also bivalent or more multivalent antibodies. The multivalent antibodies of the present invention include multivalent antibodies that all have the same antigen-binding site, or multivalent antibodies that have some or all different antigen-binding sites.

  A preferred embodiment of the antibody described in (37) above is an antibody in which no alteration has occurred in CDR. As an example, among the antibodies described in (37) above, “an antibody in which one or more amino acids in the antibody described in (1) are substituted, deleted, added and / or inserted, A preferred embodiment of the “antibody having an activity equivalent to the antibody described in the above” is “having an activity equivalent to the antibody described in (1), wherein one or more amino acids are substituted or deleted in the antibody described in (1) An added and / or inserted antibody, the amino acid sequence shown in SEQ ID NO: 9 as CDR1, the amino acid sequence shown in SEQ ID NO: 11 as CDR2, and the amino acid sequence shown in SEQ ID NO: 13 as CDR3 An antibody comprising an H chain having ". Among the antibodies described in (37) above, other preferred embodiments of the antibody can also be expressed in the same manner.

  Here, “equivalent activity” means that the antibody of interest has the same biological or biochemical activity as the antibody of the present invention. Examples of the “activity” in the present invention include an activity that specifically binds to an Aβ oligomer and does not bind to an Aβ monomer, an anti-neurotoxic activity, an Aβ amyloid fibril formation inhibitory activity, an anti-synaptic toxic activity, and an anti-memory disorder activity. be able to.

  As a method well known to those skilled in the art for preparing a polypeptide having an activity equivalent to that of a certain polypeptide, a method for introducing a mutation into the polypeptide is known. For example, those skilled in the art will recognize site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100 , 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Proc Natl Acad Sci USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766), etc. Antibodies can be prepared. Amino acid mutations can also occur in nature. Thus, an antibody having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the antibody of the present invention and having an activity equivalent to that of the antibody is also included in the antibody of the present invention. In such mutants, the number of amino acids to be mutated is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids).

  The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids with amino acids (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino groups with base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses represent single letter symbols of amino acids).

  In general, a polypeptide having an amino acid sequence modified (deleted, added and / or substituted with another amino acid) by one or more amino acid residues in a certain amino acid sequence has its biological activity (function). It is known to maintain.

  In addition to the above-described modifications, the antibody of the present invention may be further linked to other substances as long as it retains activity. Examples of other substances include peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These modifications can be made to confer additional functions or to stabilize the antibody.

  Antibodies in which a plurality of amino acid residues are added to the amino acid sequence of the antibody of the present invention include fusion proteins containing these antibodies. A fusion protein is a fusion of these antibodies and another peptide or protein. In the method for producing a fusion protein, a polynucleotide encoding an antibody of the present invention and a polynucleotide encoding another peptide or polypeptide are linked so that the frames coincide with each other, introduced into an expression vector, and expressed in a host. Any technique known to those skilled in the art can be used. Other peptides or polypeptides to be subjected to fusion with the antibody of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 His (histidine) residues 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen Fragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment and other known peptides, GST (glutathione-S-transferase), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein), etc. Is mentioned. Preparing a fusion polypeptide by fusing a commercially available polynucleotide encoding the peptide or polypeptide with a polynucleotide encoding the antibody of the present invention and expressing the fusion polynucleotide prepared thereby. Can do.

  The antibody of the present invention may vary in amino acid sequence, molecular weight, presence or absence of sugar chain, form, etc., depending on the cell to be produced, the host, or the purification method. However, so long as the obtained antibody has the same activity as the antibody of the present invention, it is included in the present invention.

  An antibody that binds to an epitope to which the antibody according to any one of (1) to (36) binds can be obtained by methods known to those skilled in the art. For example, an epitope to which the antibody of any one of (1) to (36) binds is determined by an ordinary method, and an antibody is produced using a polypeptide having an amino acid sequence contained in the epitope as an immunogen, The epitope of the antibody produced by the method can be determined, and the antibody having the same epitope as that of any of the antibodies (1) to (36) can be obtained.

  The antibodies described in (1) to (38) above include antibodies having a modified amino acid sequence, such as the above-described low molecular weight antibodies, humanized antibodies and chimerized antibodies, non-human animal antibodies, human antibodies, and other molecules. Any antibody such as a modified antibody to which a polymer (for example, a polymer such as polyethylene glycol or the like) is bonded, an antibody in which a sugar chain is modified, and the like are included.

  One preferred embodiment of the antibody in the present invention includes a modified antibody such as a chimeric antibody or a humanized antibody. In a more preferred embodiment, the variable region is a variable region derived from 2C3 antibody, 1A9 antibody, 5A5 antibody, 5A9 antibody, 4F7 antibody, 4H5 antibody, 6E4 antibody, or 6H4 antibody, and the constant region is a constant derived from human immunoglobulin. A chimeric antibody characterized by being a region, CDR is a CDR derived from 2C3 antibody, 1A9 antibody, 5A5 antibody, 5A9 antibody, 4F7 antibody, 4H5 antibody, 6E4 antibody, or 6H4 antibody, and FR is derived from human immunoglobulin Mention may be made of humanized antibodies which are FR and whose constant region is a constant region derived from human immunoglobulin. These modified antibodies can be produced using known methods.

  Since chimeric antibodies and humanized antibodies have reduced antigenicity in the human body, they are useful when administered to humans for therapeutic purposes.

  A chimeric antibody is an antibody prepared by combining sequences derived from different animals, such as a mouse antibody heavy chain, light chain variable region and human antibody heavy chain, light chain constant region antibody, etc. . Chimeric antibodies can be made using known methods (eg, Jones et al., Nature 321: 522-5, 1986; Riechmann et al., Nature 332: 323-7, 1988; and Presta, Curr Opin Struct Biol 2: 593-6, 1992). For example, first, a variable region of an antibody of interest or a gene encoding a CDR is prepared from RNA of antibody-producing cells by polymerase chain reaction (PCR) or the like (for example, Larrick et al., “Methods: a Companion to Methods in Enzymology ", Vol. 2: 106, 1991; Courtenay-Luck," Genetic Manipulation of Monoclonal Antibodies "in Monoclonal Antibodies: Production, Engineering and Clinical Application; Ritter et al. (Eds.), Page 166, Cambridge University Press , 1995, and Ward et al., "Genetic Manipulation and Expression of Antibodies" in Monoclonal Antibodies: Principles and Applications; Birch et al. (Eds.), Page 137, Wiley-Liss, Inc., 1995). The prepared gene encoding the variable region is linked to the gene encoding the constant region or framework region. Genes encoding constant regions or framework regions can be determined in the same manner as the genes encoding CDRs, or they can be prepared based on the sequence information of existing antibodies. DNA sequences encoding the chimeric product and the CDR graft product can be fully or partially synthesized using oligonucleotide synthesis techniques. For example, synthesis for oligonucleotides as described by Jones et al. (Nature 321: 522-5, 1986) can be used. Further, in some cases, site-directed mutagenesis and polymerase chain reaction techniques can be used as appropriate. The technology for oligonucleotide-directed mutagenesis of existing variable regions described by Verhoeyen et al. (Science 239: 1534-6, 1988) or Riechmann et al. (Nature 332: 323-7, 1988) The sequence can be modified, for example, to enhance the binding ability of the chimeric antibody. In addition, if necessary, use of T4 DNA polymerase, for example as described by Queen et al. (Proc Natl Acad Sci USA 86: 10029-33, 1989; WO 90/07861) Enzymatic filling in can be used.

  For example, CDR grafting techniques are known in the art ("Immunoglobulin genes", Academic Press (London), pp260-74, 1989; Michael A et al., Proc Natl Acad Sci USA 91: 969-73, 1994). . According to this technique, the CDR of one antibody is exchanged for the CDR of another antibody. Through such an exchange, the binding specificity of the former antibody changes to that of the latter antibody. Among such chimeric antibodies, those whose framework amino acids are derived from human antibodies are called “humanized antibodies (CDR grafted antibodies)”. When using antibodies for human treatment, it is preferable to use human antibodies or humanized antibodies.

  In general, a chimeric antibody consists of a variable region of a non-human mammal-derived antibody and a constant region derived from a human antibody. On the other hand, a humanized antibody consists of a complementarity determining region of a non-human mammal-derived antibody, a framework region derived from a human antibody, and a constant region.

  In addition, after producing a chimeric antibody or a humanized antibody, amino acids in the variable region (eg, FR) or constant region may be substituted with other amino acids.

  The origin of the variable region in the chimeric antibody or the CDR in the humanized antibody is not particularly limited.

  In addition, human antibody is used for the C region of the chimeric antibody and the humanized antibody.For example, as the H chain, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε are used as the L chain. Cκ and Cλ can be used. These sequences are known. In addition, the human antibody C region can be modified to improve the stability of the antibody or its production.

  The binding activity of the antibody of the present invention to the antigen (Aβ oligomer) is, for example, absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and It can be performed using immunofluorescence or the like. In ELISA, an antibody is immobilized on a plate, an antigen against it is added to the plate, and then a sample containing the desired antibody, such as a culture supernatant of a cell producing the antibody or a purified antibody, is added. Next, a secondary antibody that recognizes the primary antibody and is tagged with an enzyme such as alkaline phosphatase is added and the plate is incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, and the absorbance is measured to evaluate the antigen binding ability of the target sample. Evaluation can be performed using BIAcore (Pharmacia).

  The present invention also provides a composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

  The inventors strongly suggested that monoclonal 1A9 or 2C3 antibodies are promising candidates for therapeutic antibodies for preventing Alzheimer-like phenotypes, as described below. For example, memory loss has been shown to correspond to synaptic dysfunction caused by soluble Aβ oligomers [Klein WL, 2001, Trends Neurosci; Selkoe DJ, 2002, Science]. Excessive accumulation and deposition of Aβ oligomers can trigger a complex downstream cascade that results in Alzheimer's disease. If so, therapeutic intervention with a composition comprising the antibody of the invention and a pharmaceutically acceptable carrier as described above may be effective in blocking this pathological cascade, which is Alzheimer's. It seems possible to treat the disease.

  In the present invention, “treatment” does not necessarily need to have a complete therapeutic effect or preventive effect on an organ or tissue exhibiting a disease or a symptom due to a disease, and may be a case where it has a partial effect.

  “Treatment of Alzheimer's disease” in the present invention is to improve at least one symptom that can be caused by Alzheimer's disease. For example, improvement or suppression of cognitive dysfunction, improvement or suppression of senile plaque formation, synaptic dysfunction Examples thereof include improvement and suppression, and decrease or suppression of Aβ accumulation in brain tissue or blood. Here, “cognitive impairment” includes, for example, long-term / short-term memory impairment, object recognition memory impairment, spatial memory impairment, and associative emotional memory impairment, which are memory impairment.

  That is, the present invention provides a pharmaceutical composition or a drug comprising as an active ingredient a composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

  In the present invention, “containing the composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier as an active ingredient” means the composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier. This means that it is included as a main component, and does not limit the content.

  Examples of the pharmaceutical composition include an anticognitive impairment agent, Alzheimer's disease agent, Alzheimer's disease progression inhibitor, senile plaque formation inhibitor, Aβ accumulation inhibitor, anti-neurotoxic agent (neurotoxic neutralizer), Aβ amyloid fiber Examples thereof include formation inhibitors and antisynaptic toxicants (synaptic toxicity neutralizers).

  The pharmaceutical composition in the present invention includes a step of administering to the subject (individual) a composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier, for example, expressed as “a method for suppressing Alzheimer's disease”. You can also Other aspects include a method of suppressing cognitive dysfunction, a method of suppressing the progression of Alzheimer's disease, a method of suppressing senile plaque formation, a method of suppressing Aβ accumulation, and neutralizing (suppressing) neurotoxic activity. A method, a method of inhibiting Aβ amyloid fibril formation, or a method of neutralizing (suppressing) synaptic toxic activity can also be mentioned. Still other aspects can include methods for either or both of the prevention and treatment of cognitive dysfunction, methods for either or both of the prevention and treatment of Alzheimer's disease.

  Alternatively, it can be expressed as the use of a composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier in the production of the pharmaceutical composition.

The present invention also relates to the following compositions.
A composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier for use in either or both of prevention and treatment of cognitive dysfunction. Either or both of prevention and treatment of Alzheimer's disease A composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier for use in the present invention. A composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier for use in inhibiting Alzheimer's disease progression. A composition comprising the above-described antibody of the present invention and a pharmaceutically acceptable carrier for use in suppressing the formation of senile plaques and senile plaques. The above-described antibody of the present invention and pharmaceutically acceptable for use in suppressing Aβ accumulation. A composition containing a carrier, a composition containing the antibody of the present invention and a pharmaceutically acceptable carrier for use in neutralizing (suppressing) neurotoxic activity, and used for inhibiting Aβ amyloid fibril formation. A composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier for use in neutralizing (suppressing) synaptic toxic activity, comprising the antibody of the present invention and a pharmaceutically acceptable carrier Composition

  The drug of the present invention can be administered to humans or other animals. In the present invention, non-human animals to which a drug is administered can include mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows, monkeys, baboons, and chimpanzees. These animals are preferably animals that exhibit at least one symptom such as cognitive dysfunction, senile plaque formation, synaptic dysfunction, Aβ accumulation in brain tissue or blood, and the like.

  The antibody contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is the antibody of the present invention, and examples thereof include the antibodies described in the present specification.

When the antibody of the present invention is used as a pharmaceutical composition, it can be formulated by methods known to those skilled in the art. For example, if desired, it can be prepared into an injectable form that can be administered parenterally by making it a sterile solution or suspension in water or any other pharmaceutically acceptable liquid. . For example, the antibody to be included in the pharmaceutical composition may be transformed into an acceptable carrier or solvent, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient It can be mixed with agents, solvents, preservatives, binders and the like to provide generally acceptable unit doses required for pharmaceutical use. The term “pharmaceutically acceptable” indicates that the material is inert and includes conventional materials used as diluents or vehicles for drugs. Suitable excipients and their formulations are, for ^{example, Remington's Pharmaceutical Sciences, 16 th} ed. (1980) Mack Publishing Co., ed. Oslo et al. Which is incorporated herein by reference.

  Other isotonic solutions including saline, glucose, and adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an aqueous solution for injection. They are used with suitable solubilizers such as alcohols, specifically ethanol and polyalcohols (eg propylene glycol and polyethylene glycol), and nonionic surfactants (eg Polysorbate 80 ™ or HCO-50) can do.

  Sesame oil or soybean oil can be used as an oily liquid, and benzyl benzoate or benzyl alcohol may be used as a solubilizer together with these. Buffers (such as phosphate buffer and sodium acetate buffer), analgesics (such as procaine hydrochloride), stabilizers (such as benzyl alcohol and phenol), and antioxidants can be used in the formulation. The prepared injection solution can be filled into an appropriate ampoule.

  Administration is preferably parenteral administration, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

  The pharmaceutical composition comprises a pharmaceutically effective amount of the active ingredient (the antibody of the present invention). A “pharmaceutically effective amount” of a compound is an amount sufficient to treat and / or prevent a disorder in which the antibody of the present invention plays an important role. Examples of pharmaceutically effective amounts are, for example, when administered to an individual (patient), eg, reduce Aβ accumulation, neutralize Aβ-induced toxicity, or reduce Aβ fibril formation, thereby reducing Alzheimer's disease It may be an amount necessary to treat or prevent a disorder caused by. Reduction or neutralization is, for example, at least about 5%, 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 99%, or 100% decrease or moderate It can be sum.

  An assessment to determine such a pharmaceutically effective amount of the antibody of the present invention can be made using standard clinical protocols including histopathological diagnosis.

  The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time. Alternatively, for example, the dose can be selected in the range of 0.001 to 100000 mg / body per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art. In the animal experiments described below, the dose was selected according to the intravenous high-dose immunoglobulin therapy (400 mg / Kg) that is applicable for insurance in humans.

  The present invention also provides a method for measuring Aβ oligomers in a sample, such as Aβ40 (Aβ1-40) oligomers and Aβ42 (Aβ1-42) oligomers. Examples of the “sample” in the present invention include a sample collected from a subject. Specifically, this method includes a step of detecting Aβ oligomer contained in a sample collected from a subject using the antibody of the present invention. Measurement of Aβ oligomers in a sample is performed by, for example, a method using a sandwich solid phase enzyme immunoassay (chemiluminescence-ELISA) using chemiluminescence, immunoprecipitation with an obtained antibody, immunoblotting, flow cytometry, mass spectrometry, Methods such as immunohistochemistry can be used.

When Aβ oligomers are detected in a sample collected from a subject by the measurement method, the subject is considered to be a candidate for Alzheimer's disease. For example, when the amount of Aβ oligomer in a sample collected from each of a subject and a healthy person is compared, and it is determined that the amount of Aβ oligomer in the subject is large compared to the amount of Aβ oligomer in the healthy person, the subject has Alzheimer's disease. Judged to be a candidate. The diagnosis of whether or not a subject is a candidate for Alzheimer's disease is usually performed by a doctor (including those who have received instructions from the doctor; the same shall apply hereinafter), but each of the subject and healthy person obtained by this diagnostic method Data on the amount of Aβ oligomers in samples taken from can be useful for diagnosis by physicians. Therefore, this diagnostic method can also be expressed as a method of collecting and presenting data useful for diagnosis by a doctor.
That is, the present invention provides a method for diagnosing whether or not a subject is a candidate for Alzheimer's disease, which comprises detecting Aβ oligomers in a sample collected from the subject using the antibody according to the present invention. To do.

Further, the present invention is a method for diagnosing whether or not a subject is an Alzheimer's disease candidate comprising the following steps, and when the ratio described in step (b) is higher than that of a healthy subject, A method for determining a candidate for Alzheimer's disease is provided.
(A) A step of contacting a sample collected from a subject, the antibody according to the present invention, and an antibody that binds to Aβ monomer (b) a step of measuring a ratio of Aβ oligomer to Aβ monomer in the sample

  First, in this method, a sample collected from a subject, the antibody described in the present invention, and an antibody that binds to Aβ monomer are contacted. Here, “contact” can be performed, for example, by adding each of the above antibodies to a sample collected from a subject in a test tube. In this case, as the shape of the added antibody, a solid or the like obtained by solution or lyophilization can be used as appropriate. When added as an aqueous solution, it may be an aqueous solution containing pure antibody alone, for example, a surfactant, an excipient, a colorant, a flavoring agent, a preservative, a stabilizer, a buffering agent, a suspension. It may be a solution containing a turbidity agent, an isotonic agent, a binder, a disintegrant, a lubricant, a fluidity promoter, a corrigent and the like. The concentration of the antibody to be added is not particularly limited. For example, 500 mg, 1000 mg, 2,500 mg preparations and the like can be suitably used in the lyophilized state as with human immunoglobulin preparations.

  Next, the ratio of Aβ oligomer to Aβ monomer in the sample (in this specification, sometimes referred to as “O / M index”) is measured. For the measurement of the ratio, the following method is preferably used. For example, as described in Examples described later, it can be carried out using a method such as a ratio of oligomer and monomer ELISA values obtained from the same specimen.

  Next, this ratio is compared with the ratio in the case of a healthy person. Then, when the ratio of subjects is higher than that of healthy subjects, the subject is determined to be an Alzheimer's disease candidate.

  The diagnostic method of the present invention can be performed in vitro or in vivo, but is preferably performed in vitro.

  The “sample” in the present invention is not particularly limited as long as it is preferably a tissue derived from a subject. For example, a subject's brain (brain parenchyma etc.), an organ, a body fluid (blood, cerebrospinal fluid etc.) can be mentioned. In the present invention, blood (more preferably plasma) or cerebrospinal fluid is preferable.

  The present invention also provides a drug for use in the method for measuring Aβ oligomers in the above-mentioned sample or the method for diagnosing whether or not a subject is a candidate for Alzheimer's disease.

  In the present invention, pharmaceutical compositions comprising antibodies can be included in products and kits containing materials useful for treating a subject's pathological condition. The product can include a container of any of the compounds containing the label. Suitable containers include bottles, vials, and test tubes. The container may be formed from a variety of materials such as glass or plastic. The label on the container surface should indicate that the composition is used to treat or prevent one or more conditions of the disease. The label may also indicate instructions for administration.

  In addition to the container described above, a kit comprising a pharmaceutical composition comprising an antibody can optionally comprise a second container containing a pharmaceutically acceptable diluent. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. .

  If desired, the pharmaceutical composition may be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredients. The pack may include, for example, a metal or plastic foil such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

  In the above drugs and kits, in addition to the antibody of the present invention which is an active ingredient, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, protein stabilizer (BSA or Gelatin, etc.), a preservative, a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like may be mixed as necessary.

  The present invention has also shown that the antibody of the present invention is effective in preventing Alzheimer's disease. That is, the present invention provides a method for suppressing the progression of Alzheimer's disease, comprising the step of administering a composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier to a person suffering from Alzheimer's disease. .

  It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Method]
Production of antigen (1A9 and 2C3)
Synthetic Aβ1-42 (Peptide Laboratories, Osaka) was dissolved in distilled deionized water or 10 mM phosphate buffer, incubated at 37 ° C for 18 hours, and then applied to NuPAGE Tris-Glycine gel 4-12% SDS-PAGE. The peptides were separated and visualized by CBB staining, and then only the Aβ1-42 tetramer free of Aβ1-42 monomer contamination was excised.

Production of antigen (4F7, 4H5, 5A5, 5A9, 6E4 and 6H4)
A modified A-beta was synthesized by chemically binding the fluorescent dye 6-Carboxytetramethylrhodamine (6-TAMRA) (SIGMA) to the N-terminus of the synthesized A-beta 1-40 peptide (Peptide Institute). This modified A-beta was polymerized with a synthetic A-beta 1-40 peptide to prepare a preparation (Aβ1-40 oligomer) rich in oligomers. As a condition at this time, it is desirable that the fluorescence intensity measured using a ThT assay described later is at least 1/4 or less compared to when the modified A-beta was not mixed, more specifically, A modified A-beta and a synthetic A-beta 1-40 peptide mixed at 100 μM each and polymerized for 20 hours are preferred.

Preparation of antibody-producing hybridoma The antigen prepared by the above-described method was immunized against the meat shark of Balb-c mice, followed by six additional immunizations. Hybridomas were produced by fusion with Sp2 / O-Ag14 cells using polyethylene glycol 1500 using inguinal lymph nodes.

Antibody Isotype Determination The isotype determination of purified immunoglobulin was performed using a Serotec (Oxford, UK) mouse monoclonal antibody isotype determination kit.

Dot blot analysis (primary screening)
The initial screening was performed by dot blot analysis in which 2.5 μl of Aβ1-42 (2.5 μg / dot) preincubated for 18 hours was immobilized on a nitrocellulose membrane. Non-specific binding sites on the membrane were blocked with a phosphate buffer containing 5% low-fat milk, 1% BSA and 0.05% Tween-20, and then incubated with the culture supernatant. The Aβ oligomer-binding antibody in the culture supernatant was detected with horseradish peroxidase-labeled goat anti-mouse F (ab ') ₂ (1: 3000; Amersham) and highly sensitive chemiluminescence using LAS3000 mini (Fujitsu, Tokyo, Japan) ECL) Imaged with kit. Among 400 clones, 16 dot blot positive 16 species containing 1A9 and 2C3 were subjected to secondary screening as described below.

Immunoprecipitation and immunoblot analysis (secondary screening)
A amyloid fraction containing a large amount of Aβ oligomers (Matsubara E, et al, Neurobiol Aging) for secondary screening to examine whether the 16 clones selected in the primary screening can capture Aβ oligomers in AD brain , 2004) was conducted immunoprecipitation experiments (Ghiso J, et al, Biochem J, 1993). The buffer insoluble and formic acid soluble fraction prepared from AD brain was incubated with the culture supernatant and protein-G-Sepharose. Immunoprecipitated Aβ oligomers were separated by NuPAGE 4-12% Bis-Tris-Glycine gel, and 10 mM 3-cyclohexylamino-1-propanesulfonic acid (pH 11) containing 10% methanol, and nitrocellulose membrane Alternatively, transcription was performed at 400 mA for 1 hour on Immobilon P (Millipore). Non-specific binding sites on the membrane were blocked with phosphate buffer containing 5% low fat milk, 1% BSA and 0.05% Tween-20 for 3 hours at room temperature. Detection of immunoprecipitated Aβ oligomers was performed by immunoblotting using monoclonal 4G8 (1: 1000) or 6E10 (1: 1000) as described above, and 2 out of 16 clones, namely 1A9 and 2C3 Were selected as therapeutic antibody candidates for Alzheimer's disease.

Antibody monoclonal antibodies 6E10 and 4G8 (Covance Immuno-Technologies, Dedham, MA) recognize human Aβ sequences 1-16 and 17-24 as epitopes, respectively. Polyclonal A11 that specifically recognizes Aβ oligomers was purchased from Biosource (Camarillo, Calif.). Alex Fluor (AF) 488 or 594-conjugated goat anti-mouse IgG and Alex Fluor (AF) 488-conjugated goat anti-rat IgG were purchased from Molecular Probes (Eugene, OR). Anti-mouse IgG2b was purchased from Sigma (St. Louis, MO). Anti-synaptophysin antibodies were purchased from Santa Cruz (Santa Cruz, CA), and anti-drebrin antibodies were purchased from MBL (Nagoya, Japan).

Size exclusion chromatography (SEC)
SEC was performed to further evaluate the size specificity of 1A9 or 2C3. As previously reported [Matsubara E, et al; Neurobiol Aging, 25: 833-841, 2004], this method allows selective separation of Aβ monomers and Aβ oligomers, lipoprotein-binding Aβ and non-lipoprotein-binding Aβ. Was possible. The present inventors concentrated the culture supernatant from HEK293 cells strongly expressing APP / PS1 about 10 times using a Microcon 3 kDa molecular weight cut-off filter (Millipore Corp.), and then equilibrated with a phosphate buffer solution. 28 fractions of 1 ml each were fractionated with a Superose 12 size exclusion column (1 cm × 30 cm, Pharmacia Biotech., Uppsala, Sweden, flow rate 0.5 ml / min). Half of each fraction was immunoprecipitated with 1A9 or 2C3. Aβ present in the resulting immunoprecipitate was detected by immunoblotting using 4G8.

  Pooled cerebrospinal fluid (CSF) and lipoprotein-removed CSF from Alzheimer's disease patients and 10 age-matched healthy cases were also fractionated under the same conditions. Identification of the recovered fraction of Aβ was performed by ELISA analysis of each fraction. For the lipid evaluation, total cholesterol was enzymatically measured using a standard kit (Wako, Osaka, Japan). Under our experimental conditions, CSF lipoprotein was eluted in fractions 7-14 and fractions 15-28 contained cholesterol-free protein.

Preparation of seed-free Aβ solution After dissolving synthetic Aβ1-42 in 0.02% ammonia solution at 250 μM, the undissolved peptide that may act as a seed was prepared by Optima TL for the preparation of seed-free Aβ solution. Ultracentrifugation was performed at 540,000 xg for 3 hours using an ultracentrifuge (Beckman, USA). The supernatant was dispensed after collection and stored at -80 ° C until use. For sample preparation, an Aβ stock solution thawed immediately before use was diluted 10-fold with Tris-buffered saline (TBS: 150 mM NaCl and 10 mM Tris-HCl, pH 7.4), and a 25 μM solution was used in the following experiment. Synthetic Aβ1-40 (HCL form, Peptide Laboratories, Osaka) was adjusted at twice the concentration.

Aβ incubation and ThT assay (Yamamoto N, et al: J Biol Chem, 282: 2646-2655, 2007)
Aβ solution (25 μM) was incubated at 37 ° C. for 2 or 24 hours in the presence of the indicated concentration of antibody. The ThT fluorescence intensity in the incubation mixture was measured using a fluorescence spectrophotometer (RF-5300PC) (Shimadzu Co., Kyoto, Japan). The optimal fluorescence intensity of Aβ amyloid fibrils was measured using a reaction mixture (1.0 ml) containing 5 μM ThT and 50 mM glycine-NaOH at pH 8.5, with the excitation wavelength and emission wavelength being 446 nm and 490 nm, respectively. The fluorescence intensity was measured immediately after preparing the mixture.

  In addition, the Aβ amyloid fibril formation inhibitory activity of 4F7, 4H5, 5A5, 5A9, 6E4, and 6H4 antibodies is determined by the presence or absence of each Aβ1-42 solution diluted to a concentration of 12.5 μM in a cell culture medium. The sample was incubated at 37 ° C. for 24 hours, and then evaluated by measuring the amount of amyloid fibril formation by the ThT fluorescence intensity measurement method.

Aβ-induced neurotoxicity assay (Yamamoto N, et al: J Biol Chem, 282: 2646-2655, 2007)
Rat pheochromocytoma PC12 (PC12) cells were treated with Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated horse serum (Invitrogen) and 5% fetal bovine serum (FBS) (Invitrogen) ). In order to induce differentiation into nerve cells, PC12 cells were plated at a density of 20,000 cells / cm ² on a culture dish coated with poly-L-lysine (10 mg / ml), and 100 ng / ml nerve growth factor was added. The cells were cultured in DMEM for 6 days (PC12N) (NGF; Alornone Labs, Jerusalem, Israel). PC12N was exposed to seed-free Aβ1-42 or pre-incubated Aβ1-42 25 μM each for 48 hours at 4 ° C. in the presence or absence of antibody. Neurotoxicity caused by Aβ1-42 was assessed by Live / Dead two-color fluorescence assay according to manufacturer's instructions (Molecular Probes, Eugene, Oregon).

  Further, the Aβ-induced neurotoxicity neutralizing activity of 4F7, 4H5, 5A5, 5A9, 6E4 and 6H4 antibodies was evaluated using the following method. First, human neuroblastoma cells (SH-SY5Y) were cultured for 24 hours in DMEM containing 10% FBS at a density of 150,000 cells / well on a 24-well plate. Next, the medium was replaced with a serum-free medium containing Aβ1-42 (12.5 μM) with or without antibody added, and further cultured for 24 hours. The cytotoxicity induced by Aβ1-42 was measured by the CytoTox96 kit (Promega) based on the amount of dead cell-derived LDH released into the medium.

To characterize the size at 25 μM of ultrafiltration and molecular sieve neurotoxic Aβ oligomers, continuous ultrafiltration using Microcon 3 kDa, 10 kDa, 30 kDa, and 100 kDa cut-off membranes was performed. Four filtrates (<3 kDa, 3-10 kDa, 10-30 kDa, 30-100 kDa) and one retentate (> 100 kDa) were prepared. Each fraction was subjected to Aβ-induced neurotoxicity assay as described above. PC12N was exposed to each fraction and the toxic fraction was evaluated as described above. The distribution identification of the A11, 1A9, 2C3 or 4G8-recognizing three-dimensional structure was also performed by the dot blot analysis described above. The form of the neurotoxic oligomer was identified by subjecting each fraction to atomic force microscopy.

Electron microscopy (EM) and atomic force microscopy (AFM)
For electron microscopy, samples were diluted with distilled water and spread on a carbon-coated grid. The grid was negatively stained with 1% phosphotungstic acid and examined using a Hitachi H-7000 electron microscope (Tokyo, Japan) at an acceleration voltage of 77 kV. AFM assessment was performed as reported recently. The sample was dropped onto the newly cleaved mica. After standing for 30 minutes and washing with water, the solution samples were evaluated using a Nanoscope IIIa (Digital Instruments, Santa Barbara, CA, USA) set to tapping mode [Tero, R, et al., Langmuir 20, 7526-7531, 2004]. OMCL-TR400PSA (Olympus, Japan) was used as a cantilever.

Tissue subjects and extraction This study is based on autopsy cases (n = 50; 26 males, 24 females) by the brain bank of the Tokyo Metropolitan Institute of Gerontology (Itabashi, Tokyo, Japan). This research project has been approved by the institutional ethics committees of the University of Tokyo School of Medicine, Tokyo Metropolitan Institute of Gerontology, Tokyo Metropolitan Geriatric Medical Center, and National Institute for Longevity Medicine. Subject and sampling details have been reported but are characterized by insoluble brain fraction analysis [Katsuno T, Neurology, 64: 687-692, 2005]. In this project, the present inventors analyzed soluble brain fractions whose characteristics were not clarified in previous studies [Katsuno T, Neurology, 64: 687-692, 2005]. Frozen tissue samples (anterior portion of entorhinal cortex) were homogenized in 9 volumes of Tris-Saline (TS) buffer containing a protease inhibitor cocktail. One third (0.5 ml) of the supernatant collected after ultracentrifugation of this homogenate at 265,000 × g for 20 minutes was subjected to immunoblot analysis.

Immunohistochemistry
The left hemisphere of the brain of Tg2576 mice was cut in the sagittal direction as a 30 μm thick section using a freezing microtome (RM 2145; Leica, Wetzlar, Germany). Thioflavin-S staining was performed as previously described (Wyss-Coray et al, 2001). Tumorous neurite formation was observed using synaptophysin antibody (Chemicon, Temecula, CA). For each mouse, 4-5 slices per brain left hemisphere and thioflavin-S positive plaques and synaptophysin positive tumors were observed. The number of sexual neurites was calculated at a magnification of 40 times. For observation of Aβ deposition, serial sections were subjected to short-term pretreatment with formic acid or protease-K, followed by Aβ immunostaining kit (Sigma, St. Louis, MO), and immunopositive signals using ABC elite kit (Vector Laboratories) I drew it. Cerebral cortex and hippocampus images were captured using a digital camera connected to a microscope and analyzed using simple PCI software (Compix Imaging System, Lake Oswego, OR). The transfer of the antibody into the brain was observed using a confocal laser microscope (Carl Zeiss LSM510). Thioflavin-S positive plaques and synaptophysin positive swollen neurite counts were reported in a double-blind fashion.

Passive immunotherapy and behavioral analysis
Three month old female non-transgenic (non-Tg) mice and Tg2576 mice carrying the human APP gene with the Swedish double mutation of familial AD (K670N; M671L) and overexpressing Tac2576 mice (Germantown, NY, USA) and was reared in the animal breeding facility of the present inventors until the age of 13 months. To determine if passive immunotherapy prevents the Alzheimer-like phenotype, 4 months Tg2576 is tailed with 1A9 or 2C3 (0.4 mg / kg / week) or PBS until 13 months of age. It was administered intravenously. Memory function was measured at the age of 13 months with respect to four behavioral paradigms as previously described (Mouri A, FASEB J, 21: 2135-2148, 2007): (1) Short-term memory in the Y maze test; (2) New object recognition test; (3) Morris water maze test; (4) Fear conditioning test with clues. Mice were sacrificed for biochemical and histological evaluation 3 days after the end of the behavioral test. The experimental results were analyzed by one-way ANOVA and two-way ANOVA, and Fisher test was used for post-hoc analysis.

Lipoprotein separation and removal
After separation of CSF, continuous density flotation for preparation using 600 μl CSF and previously described protocol [Matsubara E, et al; Ann Neurol, 45: 537-541, 1999] in 12 AD patients and 13 NC patients Lipoprotein was removed by ultracentrifugation. The density of CSF was adjusted to 1.25 g / ml with KBr, and ultracentrifuged at 100,000 rpm and 16 ° C. for 8 hours using a Hitachi RP100AT centrifuge. Both suspended lipoprotein and density-removed supernatant (LPD-CSF) at a density of 1.25 g / ml were subjected to ultrafiltration using a 3 kDa cutoff membrane (Microcon 3; Arnicon, Inc) and frozen until use Stored below or at 4 ° C.

  Furthermore, lipoproteins were also removed by affinity chromatography using PHML-LIPOSORB (Calbiochem, La Jolla, Calif.). After mixing for 60 seconds at a ratio of PHML-LIPOSORB (Calbiochem, La Jolla, CA) 1 to sample (plasma or brain) 1.5, the supernatant (lipoprotein-removed sample) obtained by centrifugation at 3,000 rpm for 10 minutes, It was subjected to 6E10 oligomer ELISA. Elution of the lipoprotein-bound sample bound to PHML-LIPOSORB was performed with 20 mM sodium deoxycholate. Specific lipoprotein removal was confirmed by FAST-RED 7B (Wako, Osaka, Japan) staining after 1% agarose gel electrophoresis (Beckmann).

Human Aβ quantification as previously described in detail [Matsubara E, et al, Ann Neurol, 537-541, 1999; Matsubara E, et al, Neurobiol Aging, 25: 833-841, 2004], whole plasma or A sandwich ELISA was used to specifically quantify LPDP Aβ species. For analysis of brain Aβ species, 100 μl of buffer soluble Aβ species is directly applied to the ELISA, and an insoluble Aβ sample in the form of 70% formic acid extract is washed with 1 M Tris-HCl (pH 8.0) prior to ELISA. And diluted 1: 1000. The resulting value was corrected by brain wet weight and was finally expressed as pmol / gram. Plates were normalized to each other by including three standard plasma samples on all plates.

ELISA specific for Aβ oligomers
In order to specifically detect Aβ oligomers but not monomeric Aβ, a sandwich solid phase enzyme immunoassay (chemiluminescence-ELISA) utilizing chemiluminescence was used. Microplates were coated with a mixture of monoclonal 1A9 (IgG2b isotype), 2C3 (IgG2b isotype) or 1A9 / 2C3 and incubated with 100 μl of different samples (brain and cerebrospinal fluid) at 4 ° C. for 24 hours. Peroxidase-conjugated BA27 Fab 'fragment (anti-Aβ1-40, specific for Aβ40, Wako pure chemical, Osaka, Japan) or horseradish peroxidase-conjugated BCO5 Fab' fragment (anti-Aβ35-43, specific for Aβ42, Wako pure chemical, Osaka, Japan) and incubated at 4 ° C. for 24 hours. Chemiluminescence generated using SuperSignal ELISA Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) was quantified with a Veritas microplate luminometer (Promega).

  To evaluate the efficacy of peripheral administration of monoclonal 1A9 or 2C3 in vivo, Aβ oligomer analysis in plasma and organs obtained from treated mice was performed using 6E10-HRP-labeled 6E10 (Senetek PLC, Napa, CA, USA) humans. A specific oligomer ELISA was used. In order to increase sensitivity, detection was performed by the chemiluminescence system described above. In order to avoid detection of lipoprotein-binding Aβ monomer, the present inventors used a sample obtained by removing lipoprotein from plasma or organ in advance using PHML-LIPOSORB as described above.

Inhibition ELISA
The Aβ oligomer used in this assay was prepared by diluting synthetic Aβ1-40 (HCl form) to a concentration of 0.1 mg / ml with PBS and incubating at 37 ° C. for 1 hour. The Aβ monomer was prepared by diluting synthetic Aβ1-40 (TFA form) with PBS to a concentration of 0.1 mg / ml. First, 400 ng / well of Aβ oligomer was immobilized on a 96-well-immuno-plate, followed by blocking using BSA. Next, 4F7, 4H5, 5A5, 5A9, 6E4 and 6H4 antibodies and anti-Aβ antibodies (4G8 and 6E10) as controls were serially diluted in the range of 100 pg / ml to 100 μg / ml. After incubation for a period of time, each was added to the 96 well-immuno-plate and incubated for 10 minutes at room temperature. The binding force between the solidified Aβ oligomer and each antibody was detected by measuring the absorbance at 450 nm using a color reaction using an HRP-labeled anti-mouse IgG antibody and a TMB solution.

[Example 1] Production of Aβ oligomer-specific monoclonal antibodies (1A9 and 2C3) In the solution, Aβ oligomers and monomers coexist. For this reason, removal of Aβ monomer is indispensable in the preparation of an antigen for obtaining an Aβ oligomer-specific antibody. As shown in FIG. 1A, the present inventors were able to isolate only the SDS-stable Aβ tetramer without contamination of Aβ monomer by SDS-PAGE analysis. For selection of positive hybridoma clones after in vivo immunization with the isolated Aβ tetramer, a two-stage screening was performed in which dot blot analysis was performed followed by immunoprecipitation. Of 400 clones subjected to dot blot analysis, 16 were determined to be positive (positive rate = 4%). To assess the oligomer specificity of selected positive clones, an amyloid fraction derived from AD brain that is insoluble in phosphate buffer and soluble in formic acid (FA) [Matsubara E et al, Neurobiol Aging, 25: 833-841 , 2004] was analyzed by immunoprecipitation using the supernatant of a positive hybridoma cell culture (FIG. 1B). Immunoblot analysis with anti-Aβ monoclonal 4G8 detected dimers, smaller trimers, and higher molecular weight smears characteristic of aggregated Aβ molecular species. Aβ monomer dissociated in the presence of a small amount of SDS was also observed. In order to further verify the presence of Native 1A9 or 2C3 conformations (oligomers), the present inventors in conditioned medium (CM) of human fetal kidney (HEK) 293 cells transfected with mutant PS1 cDNA. Detection was aimed at (Nakaya Y et al, J Biol Chem, 280: 19070-19077, 2005). The present inventors tried to identify oligomers after fractionating HEK293 CM using SEC. As reported previously (Matsubara E et al, Neurobiol Aging, 25: 833-841, 2004; Yamamoto N, et al: J Biol Chem, 282: 2646-2655, 2007) 13) is effectively separated from the monomer (fractions 14 to 20). Monoclonal 1A9 contains SDS-stable Aβ dimers secreted into CM in fraction 8 (> 680 kDa), SDS-stable dimers and trimers in fraction 13 (17-44 kDa), and very small amounts of dimer. Was immunoprecipitated in fraction 16 (FIG. 1C). Similar results were obtained with immunoprecipitation with 2C3 (data not shown). These data clearly showed that monoclonal 1A9 or 2C3 is truly specific for Aβ oligomers without recognizing Aβ monomers.

[Example 2] Anti-neurotoxic activity of monoclonal 1A9 and 2C3 To evaluate whether monoclonal 1A9 or 2C3 can protect against Aβ-induced neurotoxicity, PC12 cells (PC12N) differentiated with NGF were treated with monoclonal antibodies. Incubated with seed free 25 μM Aβ 1-42 (ThT negative supernatant with 540,000 × g) for 48 hours at 37 ° C. in the presence or absence of (mAb). Neuronal viability was determined using the LIVE / DEAD assay (Figure 2). Compared to the control assay (FIG. 2A), significant neuronal cell death (50%) was observed in the presence of Aβ1-42 (FIGS. 2B and 2G). Nonspecific IgG2b (FIGS. 2C and 2G) was unable to inhibit Aβ1-42-induced neurotoxicity under the same conditions. The commercial Aβ-specific monoclonal antibody 4G8 (IgG2b isotype, FIGS. 2D and 2G) tended to enhance toxicity. Monoclonal 2C3 (IgG2b isotype, Figures 2F and 2G) neutralized Aβ1-42 neurotoxicity almost completely in a concentration-dependent manner, so the de novo-forming neurotoxic 2C3 conformation takes oligomeric form. It was thought that there was. On the other hand, the anti-neurotoxic activity of 1A9 (IgG2b isotype, FIGS. 2E and 2G) is intermediate between 2C3 and non-specific IgG2b, indicating that the 1A9 conformation has a structurally different conformation from the 2C3 oligomer. I suggest that.

[Example 3]
The identification of the exact size and morphology of neurotoxic Aβ1-42 oligomers is currently one of the most important challenges and is under intense competition. We were able to isolate the soluble neurotoxic Aβ1-42 molecular species by ultrafiltration and molecular sieve (UC / MS) and split it into the following 5 fractions (FIG. 3A): fraction 1, < 3 kDa filtrate (lane 1); fraction 2, 3-10 kDa filtrate (lane 2); fraction 3, 10-30 kDa filtrate (lane 3); fraction 4, 30-100 kDa filtrate (lane 3) Lane 4); fraction 5,> 100 kDa retentate (lane 5). By immunoblot analysis using monoclonal 4G8 (Figure 3A), there was no Aβ in fraction 1 (lane 1), Aβ monomer in fraction 2 (lane 2), and Aβ monomer in (lane 3). A small amount of dimer, Aβ from monomer to pentamer in fraction 4 (lane 4), and 45 to 160 kDa band from monomer to pentamer in fraction 5 (lane 5) It was. These data indicate that 2% SDS depolymerizes high molecular weight (HMW) Aβ oligomers into monomers and low molecular weight (LMW) oligomers. In order to verify the size distribution of toxic Aβ1-42, we examined the biological activity of each fraction co-incubated with PC12N at 37 ° C. for 48 hours. As shown in FIGS. 3B and 3C, fraction 1 was non-toxic, fraction 2 had negligible toxicity, indicating that Aβ monomers and dimers are unlikely to be toxic. . Fractions 3-5 showed significant toxicity [One-way ANOVA analysis, p <0.0001], suggesting that the size of neurotoxic oligomers is theoretically equivalent to or larger than trimers. In the dot blot analysis using the oligomer-specific A11 antibody, A11 positive findings were observed in the above three fractions (3-5) having neurotoxicity, and the evidence that the neurotoxic substance was indeed an oligomer was obtained ( (Figure 3D). 2C3 oligomers were observed in fractions 4 and 5 (FIG. 3D), confirming that 2C3 actually reacts with neurotoxic Aβ oligomers (> 30 kDa). Furthermore, most of the 2C3 oligomers were found in the most toxic fraction 5 (> 100 kDa), and 2C3 oligomers with molecular weights above 100 kDa were considered to exhibit strong neurotoxicity (Figure 3D). On the other hand, the 1A9 oligomer is slightly distributed in the most toxic fraction 5 but very small amount, which is in good agreement with the result of insufficient neurotoxic neutralizing activity by 1A9 (FIGS. 2E and 2G). In contrast, monoclonal 4G8, which has no anti-neurotoxic activity, detected Aβ species distributed in all fractions (Fig. 3D), and oligomers may exist as non-toxic polymers even at the same size. It was.

  Each fraction was subjected to atomic force microscopy (AFM) to further verify the toxicity-structure relationship. Only three fractions exhibiting neurotoxicity revealed a spherical particle morphology corresponding to the fraction size. Figure 3E shows interatomic intervals between non-toxic fraction 2 (Fr.2), toxic fractions 3 (Fr.3) and 4 (Fr.4), and the most toxic fraction 5 (Fr.5). A force microscope image is shown. It is clear that countless granular polymer molecules are formed in the toxic fraction, and in particular, fraction 5 is composed of heterogeneous toxic molecules mixed with bead-shaped molecules and ring-shaped molecules in addition to large and small granular molecules. It became.

[Example 4] Aβ amyloid fibril formation inhibitory activity of 1A9 or 2C3 Next, the inventors examined whether 1A9 or 2C3 has Aβ amyloid fibril formation inhibitory activity. Aβ1-42 amyloid fibril formation (0, 10, 25, 50 μM) was assayed by ThT fluorescence for 72 hours at 37 ° C. Under the conditions enforced by the present inventors, seed-free Aβ1-42 (ThT-negative supernatant fraction after ultracentrifugation at 540,000 × g) was polymerized into amyloid fibrils by a mechanism dependent on polymerization nuclei (Fig. 4A). To assess the inhibitory activity of 1A9 or 2C3 on Aβ amyloid fibril formation, we incubated 25 μM seed-free Aβ1-42 at 37 ° C. for 48 hours in the presence or absence of antibody. As shown in FIG. 4B, the change in ThT fluorescence intensity was dependent on the concentration of 2C3, but no change was observed in the presence of monoclonal 1A9, 4G8 or non-specific IgG2b. On the other hand, in the polymerization of Aβ after 2 hours of incubation, almost complete fibrosis inhibitory activity was observed in 1A9 as well as 2C3 (FIG. 4C). Aβ amyloid fibril formation inhibitory activity is observed even when the molar ratio of 2C3 to Aβ is low, and it is estimated that 2C3 has an inhibitory effect on polymerization nuclei or seed function formed de novo early in Aβ1-42 amyloid fibril formation It was done. Similar results were observed morphologically. As shown in Fig. 4E (Aβ42 alone) and Fig. 4F (Aβ42 + 2C3, 25: 3), Aβ amyloid fibril formation was partially inhibited in the presence of monoclonal 2C3, and only slightly inhibited in the presence of 1A9 (Fig. 4G). Was confirmed by electron microscopy (EM). On the other hand, none of the test antibodies was observed to dissolve or depolymerize Aβ1-42 amyloid fibrils formed in advance by incubation for 24 hours (FIG. 4D).

[Example 5] Toxicity-related oligomers targeted by 1A9 or 2C3
To elucidate the structural and kinetic relationship between Aβ1-42 oligomerization and amyloid fibril formation, the time course of polymer formation was performed by dot blot analysis using A11, 1A9, 2C3 or 4G8 It was. As shown in FIG. 5A, most of the A11 antibody-reactive oligomers are constructed during the lag time period (0 to 8 hours) of polymerization, and the ThT-fluorescence values are relatively weak. In the next fiber elongation phase (8-24 hours), the level of A11 immunoreactive oligomers reached a plateau and then remained constant for 72 hours (plateau phase) (around 20% of the peak value) . Since the anti-oligomer A11 antibody does not recognize amyloid fibrils and has been shown to specifically observe Aβ oligomer formation [Kayed R, et al, Science 300, 486-489, 2003], the present inventors These results suggest that there is an oligomeric polymerization state in which Aβ oligomerization precedes amyloid fibril formation and does not directly transfer to the amyloid fibril formation pathway. The kinetics of 2C3 and A11 oligomers were similar, but the kinetics of 1A9 oligomers were different. The 1A9 oligomer was detected for the first time after 4 hours, and then the 1A9 immunoreactivity increased by a factor of about 2 over time. This suggests that the 1A9 oligomer takes delayed formation for the time being. On the other hand, 2C3 oligomers must be transiently present in the lag time period (O to 8 hours) and then remain in a very small amount (less than 5%) as an oligomer polymerization state for 8 to 72 hours. Became clear. These data from the inventors show that A11, 1A9 and 2C3 oligomers have structurally and immunologically different conformations or stability, and 2C3 oligomers can be relatively unstable compared to 1A9 oligomers. Sex was suggested.

  To investigate De novo's toxic polymerization status, seed-free Aβ1-42 (0 hour) and Aβ1-42 (Figure 5B) preincubated for 2, 4, and 24 hours were exposed to PC12N for 48 hours at 37 ° C. And its neurotoxic activity was verified. As shown in FIG. 5B, immunoblot analysis using 4G8 revealed that monomer to trimer already existed at time zero. In the 2 hour and 4 hour preincubations, a high molecular weight (HMW) smear pattern with a molecular weight of 45-160 kDa was obtained in addition to the pentamer from the monomer. The amount of HMW smear decreased dramatically at 24 hours, and the main components were two groups: a polymer group that remained in the well and could not enter the gel, a dimer, and a small amount of monomer. The HMW smear disappeared after 48 hours of incubation at 37 ° C. As shown in FIGS. 3A and 5C, seed-free Aβ1-42 was found to exhibit maximum toxicity at molecular weights> 100 kDa from molecular sieving experiments. It is clear on SDS-PAGE that this toxic molecule is composed of molecules exhibiting a high molecular weight (HMW) smear pattern with a molecular weight of 45-160 kDa in addition to monomer to pentamer. It has been found that low molecular weight undergoes depolymerization. However, preincubation treatment with seed-free Aβ1-42 for 2, 4 and 24 hours reduced the neurotoxic activity of Aβ oligomers formed in de novo by about 12.5%, about 26% (FIG. 5C). This result shows that the amount of Aβ oligomers formed de novo early in Aβ polymerization is a determinant of neurotoxic activity, there is a peak of formation during 0-2 hours, and the amount of formation decreases with time It was thought to be. On the other hand, there is a possibility that Aβ amyloid fibril de novo polymerization nucleus or amyloid fibril itself has a neurotoxic neutralizing effect. The present inventors removed Aβ polymerized nuclei or amyloid fibrils that had been insolubilized from Aβ1-42 incubated for 2 hours by ultracentrifugation at 540,000 × g for 3 hours. Since the 540,000 × g supernatant and pellet obtained as a result of ultracentrifugation exhibited the same level of thioflavin-T signal, ThT positive soluble Aβ polymer was 540,000 × g supernatant (ThT binding was not fibril formation but β This suggests that there is a structural change rich in the sheet structure). When only this soluble polymer was exposed to PC12N, the neurotoxicity was recovered and enhanced (FIG. 5D), suggesting that the insolubilized Aβ1-42 itself exhibited antitoxic activity. Under these conditions, monoclonal 1A9 completely neutralized the neurotoxicity induced by soluble Aβ oligomers rich in β sheet structure, exceeding the neutralizing activity of 2C3. On the other hand, non-specific IgG2b by itself does not affect the survival of PC12N in culture. Thus, the neurotoxic 1A9 polymer is essentially a more structurally modified and slightly stabilized soluble toxic oligomer, and the neurotoxic 2C3 polymer is essentially the very structural change in the early stages of the polymerization process. It is considered to be a short-lived oligomeric intermediate rich in instability.

[Example 6] Monoclonal 1A9 or 2C3 recognizes Aβ oligomers in brain parenchyma
Given the demonstrated specificity and biological activity of 1A9 or 2C3, the inventors subsequently aimed to detect 1A9 or 2C3 polymers in the brain by immunohistochemistry. The inventors first performed a conventional immunohistochemical method: formaldehyde fixation of brain sections and enhancement of immunoreactivity by formic acid, SDS or microwave treatment, and the like. No immunoreactivity was observed in either AD brain by both antibodies using either enhancement method. We pre-treated sections with protease-K, which is known to improve immunostaining [Wrzolek MA, et al: Am J Pathol, 141: 343-355, 1992] and found many senile plaques. Was immunostained with 1A9 (Figure 6A), 2C3 (Figure 6B) and A11 (Figure 6C). Combined with our in vitro experiments finding that Aβ amyloid fibrils neutralize Aβ oligomer-induced neurotoxicity, senile plaques are a kind of protective reservoir for sequestering and storing Aβ oligomers. It was thought that antibodies could not be easily reached. In fact, in the immunoprecipitation using 1A9 or 2C3, the presence of Aβ oligomers recognized by both antibodies was confirmed in the amyloid fraction composed of senile plaques, and our hypothesis is that the in vivo consistency is Proven (see Figure 1B).

  To further verify the presence of “soluble” 1A9 or 2C3 polymers in the brain, we performed immunoprecipitation studies using both antibodies. To avoid chemical modification during the extraction of soluble oligomers, brain homogenates were prepared using Tris-buffered saline (TBS). In the TBS sample, 1A9 immunoprecipitated oligomers with molecular weights corresponding to 4-mer, 5-mer, 8-mer and 12-mer from AD brain cerebral cortex (FIG. 6D, lane 2). In the healthy control brain, it was lower than the detection sensitivity (lane 3). 1A9 (lane 2) is a mixture of monoclonal 4G8 / 6E10 (lane 1), even though 1A9 and 4G8 / 6E10 have similar 4-mer, 5-mer and 8-mer intensities Seemed to recover 12-mer more strongly. Nearly identical results were obtained by 2C3 immunoprecipitation (FIG. 6D, lanes 4-6). For this reason, the inventors next attempted to identify molecules responsible for in vivo neurotoxicity found in the human entorhinal cortex. In the general elderly population, it is well known that neurofibrillary tangles (NFT) and neuronal loss precede senile plaque formation in this lesion. We hypothesized that the lack of functional reservoirs such as senile plaques for 1A9 or 2C3 polymers is detrimental to neurons in the entorhinal cortex and possibly the cause of memory impairment. Previously reported 50 autopsy cases (Katsuno et al: Neurology, 64: 687-692, 2005): AD 2 cases, Braak NFT stage I-II 35 individuals and NFT stage III-IV 13 cases, The level of 12-mer in the buffer soluble fraction was assessed by immunoblotting using monoclonal 1A9 or 2C3. As shown in FIGS. 6E (1A9) and 6F (2C3), the relative immunoreactivity intensity of 1A9- or 2C3-immunoreactive 12-mer against actin is the healthy control group (Braak NFT stage I-II). And AD patients were significantly higher than the mild cognitive impairment group (Braak stage III-IV). Interestingly, in the entorhinal cortex of healthy controls (Braak NFT stages I-II) and mild cognitive impairment (Braak stages III-IV), the 12-mer levels were approximately 45% and 60%, respectively (AD cases (As shown in Figure 6E and F). This result suggests that 12-mer accumulation precedes the emergence of cognitive dysfunction and increases as the Braak NFT stage progresses, suggesting that 1A9 or 2C3 immunoreactive 12-mer is responsible for in vivo neurotoxicity. It is suggested that it is a polymer.

[Example 7] Monoclonal 1A9 or 2C3 recognizes Aβ oligomers in cerebrospinal fluid Aβ polymer (soluble 1A9- or 2C3-immunoreactive 12-mer) responsible for in vivo neurotoxicity is present in brain parenchyma Therefore, the present inventors speculated that the same polymer would also be present in CSF. To verify this prediction, we fractionated CSF pooled with 10 patients each with AD patients and age-matched healthy controls by SEC, and each fraction was isolated to the same monoclonal in the capture and detection system. A sandwich ELISA specific for Aβ oligomers using BCO5 or BA27 was used. The BC05-BC05 oligomer ELISA identified the presence of soluble Aβ1-42 in fraction 13, while the BA27-BA27 ELISA detected the presence of soluble Aβ1-40 in fractions 7-14 (data not shown). However, the absorbance values (OD450 nm) obtained by any ELISA remained low, and there was no lack of sensitivity to detect trace amounts of Aβ oligomers present in CSF. In addition, from the examination of Aβ monomer by BNT77-ELISA in the same fraction, lipoprotein binding Aβ monomer (fractions 7-14) and lipoprotein non-binding Aβ monomer (fractions 15-17) are in the same fraction. Co-existence with Aβ oligomers was confirmed (FIGS. 7-1A and 7-1B) [Matsubara E, et al; Neurobiol Aging, 25: 833-841, 2004]. The value of lipoprotein-binding Aβ monomer in AD is similar to that of healthy controls, whereas non-lipoprotein-bound Aβ40 monomer (Figure 7-1A) and Aβ42 monomer (Figure 7-1B) are age-matched normal AD was lower than control. In these assays, we also used BCO5 or BA27 antibodies as capture antibodies, and in ELISA constructed with the same antibody labeled with HRP as the capture antibody, in addition to oligomers, they were present separately on lipoproteins. It was discovered that Aβ monomer can be detected. In previous reports using similar assays described herein (eg, 6E10 / 6E10 ELISA) [Lee EB, et al, J Biol Chem, 281: 4292-4299, 2006], this problem was overlooked. It was. Since these lipoprotein-binding Aβ monomers and oligomers are eluted with the same retention time in SEC, they cannot be distinguished from each other in an oligomer ELISA that uses the same antibody for capture and detection. To analyze Aβ oligomers with non-selective antibodies, CSF itself containing lipoprotein was found to be inappropriate as a specimen.

  In order to overcome these weaknesses of the prior methods, the present inventors devised a sample to be used and an ELISA detection antibody. As a measurement sample, CSF from which lipoprotein was removed in advance (LPD-CSF) was used, and Aβ oligomer-specific 1A9 and 2C3 were used as ELISA detection antibodies. In addition, a chemiluminescence ELISA was developed to improve sensitivity. Pooled LPD-CSF (FIGS. 7-1C to D) was fractionated by SEC, and Aβ oligomer distribution was analyzed by luminescence ELISA using 1A9 and 2C3 as detection antibodies for each fraction. As shown in Figures 7-1C-D, the presence of Aβ oligomers was identified in SEC fractions 12-15 (relatively large Aβ in the molecular weight range of 18-108 kDa, corresponding to 4-mer-24-mer). It was. In all identified fractions, levels of 1A9 · 2C3 oligomers were elevated in AD patients. In order to confirm the usefulness as a diagnostic marker, the amount of LPD-CSF Aβ oligomers was compared between AD patients and healthy controls matched in age, although it was limited to a few cases. As shown in FIG. 7-2G, the 2C3 oligomer consisting of Aβx-42 was significantly increased in AD patients compared to the normal control group (nonparametric analysis: p = 0.0103). In contrast, the 2C3 oligomer consisting of Aβx-40 showed no significant difference between the two groups. On the other hand, 1A9 oligomer consisting of Aβx-42 showed no statistically significant difference, but it tended to increase in AD patients. There was no significant difference between the two groups of 1A9 oligomers consisting of Aβx-40 (FIG. 7-2E). The structural change from Aβ monomer to oligomer is the first event in the Aβ polymerization process, and the ratio of Aβ oligomer to Aβ monomer [O / M index] corresponds to a clinical index that reflects the pathological state of AD . As shown in FIGS. 7-2F and 7-2H, the O / M index in Aβ42 or Aβ40 was significantly increased in the AD patient group compared to the healthy control group (1A9: Aβ42, P = 0.0137; Aβ40, p = 0.0429 vs 2C3: Aβ42, P = 0.0012; Aβ40, p = 0.0051). From the above results, 1A9 or 2C3 positive conformations were not only present as Aβ oligomers in LPD-CSF, but also increased in AD patients. Our results further show that the structural conversion of lipoprotein non-binding soluble Aβ to oligomeric intermediates occurs in the CSF of AD, and that detection is useful in the diagnosis of sporadic AD. It was clarified that it could be a target marker.

[Example 8] Passive immunotherapy with monoclonal 1A9 or 2C3 prevents the development of memory impairment in Tg2576
In order to verify the in vivo prophylactic treatment effect of passive immunotherapy by administration of 1A9 (n = 13) and 2C3 (n = 11), the present inventors used 1A9, 2C3 (0.4) from 4 months to 13 months of Tg2576 mice. mg / kg / week) or PBS was administered via the tail vein. The memory function at 13 months of age was determined for four learning and behavioral paradigms: (1) Short-term memory in the Y maze test (Figure 8A); (2) Object recognition memory in the new object recognition test (Figure 8B); 3) Spatial memory in the water maze test (Figure 8C); (4) Association emotional memory in the contextual fear learning test (Figure 8D). Tg2576 mice administered with PBS developed significant learning / behavioral disorders compared to Tg2576 mice administered with 1A9 or 2C3 (FIGS. 8A to 8D). Unlike Tg2576 mice treated with PBS (n = 10), the memory function of Tg2576 mice treated with 1A9 or 2C3 is indistinguishable from results obtained from a cohort of untreated wild-type mice of the same age From these results, it was confirmed that the Tg2576 mice administered with 1A9 or 2C3 maintained both the short-term memory and the long-term memory that were originally reduced in the PBS-administered group. In other words, if passive immunotherapy targeting Aβ oligomers was performed before the onset of memory impairment, it was possible to obtain evidence that it was possible to prevent the onset of memory impairment, that is, the onset of AD. This is also the first in vivo evidence that directly shows that Aβ oligomers are memory impairment-causing molecules.

Example 9 Monoclonal 1A9 prevents Aβ accumulation in Tg2576 brain
Tg2576 mice (1A9 administration group n = 13, 2C3 administration group n = 11) treated with passive immunization from 4 to 13 months old and Tg2576 mice (n = 10) treated with PBS After discontinuation, the brain (cerebral cortex vs hippocampus) Aβ accumulation amount was divided into three fractions of protease inhibitor-containing Tris buffer, soluble (2% SDS) and insoluble amyloid (2% SDS insoluble 70% formic acid soluble) 150 mg / each extract) was continuously extracted and verified. Non-accumulating physiological Aβ molecules in Tris buffer, Aβ solubilized with 2% SDS include diffuse senile plaques before amyloid fibril formation, immunocytochemically undetectable Aβ and three-dimensional structures It is thought to contain an accumulative soluble oligomer Aβ that has changed. Aβ was fractionally quantified by ELISA specific for each of Aβ40 stump and Aβ42 stump (BNT77 / BA27 specific for Aβ40, BNT77 / BC05 specific for Aβ42, WAKO kit). The Aβ concentration in the Tris buffer mainly composed of non-accumulating physiological Aβ molecules was not significantly different among the three groups (FIGS. 9A, C, Aβx-40; FIGS. 9B, D, Aβx-42). In the soluble brain Aβ accumulation amount (SDS fraction), significant accumulation inhibitory effects of cerebral cortex Aβx-40 and Aβx-42 were confirmed only in the 1A9 administration group (Fig. 9E, Aβx-40; Fig. 9F, Aβx -42). In the hippocampus, no accumulation inhibitory effect was observed (FIG. 9G, Aβx-40; FIG. 9H, Aβx-42). On the other hand, in the insoluble brain Aβ accumulation amount (FA fraction), a significant accumulation inhibitory effect of cerebral cortex Aβx-40 was confirmed only in the 1A9 administration group (FIG. 9I, Aβx-40; FIG. 9J, Aβx-42) . In the hippocampus, accumulation suppression effect was not recognized (FIG. 9K, Aβx-40; FIG. 9L, Aβx-42). In the A11 immunoblot analysis of the soluble SDS fraction, the effect of suppressing the accumulation of A11 positive oligomer (4-mer) in the cerebral cortex was confirmed in both antibody treatment groups (FIG. 9M).

[Example 10] Plasma Aβ oligomers increase in 1A9 and 2C3 passive immunotherapy
Plasma between 3 groups of Tg2576 mice (1A9 administration group n = 13, 2C3 administration group n = 11) and Tg2576 mice (n = 10) treated with PBS from 4 months to 13 months of age There was no significant difference in the intermediate Aβ concentration (FIG. 10A, Aβx-40; FIG. 10B, Aβx-42). There was no significant difference in Aβ40 / 42 ratio (FIG. 10C).

  Next, to elucidate the mechanism of expression of the preventive effect of 1A9 or 2C3 passive immunization (IVIg) against AD-like phenotype in Tg2576 mice, the present inventors used physiologically soluble and insoluble Aβ oligomers using pooled brain homogenates. Existence and the presence of Aβ oligomers in the periphery and plasma were verified. There was no change in the amount of saline soluble Aβ oligomers of brain homogenates pooled within each treatment group (FIG. 10D). On the other hand, the amount of insoluble Aβ oligomer was confirmed to decrease in the 1A9 or 2C3 treatment group (FIG. 10E). In addition, Aβ oligomers in plasma pooled within each treatment group (albumin-removed plasma, upper panel F; albumin / lipoprotein-removed plasma, lower panel F) were verified by A11 dot blot. The presence of the oligomer was confirmed (FIG. 10F). In the passive immunotherapy group, an increase in the plasma of A11 positive oligomers was evident compared to the PBS administration group (FIG. 10F). In addition, 2C3 oligomers were present in a higher proportion in the lipoprotein-binding form than 1A9 oligomers (lower panel F). Furthermore, as a result of examining Aβ oligomers in plasma by the A11 immunoprecipitation method, the oligomers of about 200 kDa increased in Tg2576 mice that received passive immunotherapy compared to the PBS administration group (FIG. 10G). The increase in plasma Aβ oligomers in this passive immunotherapy group can be considered as a result that directly reflects the increase in clearance from the brain. The direct target molecule of intravenous passive immunization is also present in blood in addition to the brain, and it is proved that oligomer selective clearance can be increased from the brain with the peripheral site of action as the clinical point of intravenous passive immunization. Consistency was confirmed.

[Example 11] 1A9 and 2C3 passive immunotherapy suppresses senile plaque amyloid and dilated neurite formation In the passive immunotherapy group, immunohistochemical Aβ deposition was suppressed (FIG. 11A). The number of thioflavin-S staining-positive senile plaque amyloid was also significantly suppressed in both cerebral cortex and hippocampus (FIG. 11B, upper), and the decrease was also histologically evident (FIG. 11B, lower). Synaptophysin positive tumor neurite formation was also significantly suppressed in the passive immunotherapy group (FIG. 11C).

[Example 12] Immunostaining analysis with anti-synaptophysin antibody or drebrin antibody
1A9 and 2C3 suppressed pre and post synaptic degeneration in the cerebral neocortex (FIG. 12).

[Example 13] Antibody migrates into the brain As a result of examining the presence and localization of Aβ deposits and mouse IgG in the brain with a confocal laser microscope, the presence of diffuse senile plaques was almost independent of Aβ deposits. The presence of mouse IgG was confirmed. This mouse IgG was observed only in the passive immunotherapy group [1A9 (FIG. 13A); 2C3 (FIG. 13B)], but not in the PBS administration group (FIG. 13C). Was thought to have moved into the brain. This result shows that the antibodies that migrated into the brain directly neutralize the toxicity of the soluble Aβ polymer, and also cleared into the blood in the form of a complex of the antibody and soluble Aβ polymer. The therapeutic effect was thought to be based on a complex mechanism of action.

[Example 14] Preparation of Aβ oligomer-specific monoclonal antibody (5A5, 5A9, 4F7, 4H5, 6E4, 6H4) and dot blot analysis Among 33 clones prepared by the above method using Aβ1-40 oligomer as an antigen, dot When confirmed by blot analysis, six types of monoclonal antibodies that specifically recognize Aβ oligomers were confirmed. When antibody isotype determination of these 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4 and 6H4) was performed, the isotype was 4F7 (κ for L chain, IgG2a for H chain), 4H5 (κ for L chain, κ, H chain is IgG2a), 5A5 (L chain is κ, H chain is IgG2b), 5A9 (L chain is κ, H chain is IgG2b), 6E4 (L chain is κ, H chain is IgG1), 6H4 (L chain is The κ and H chains were IgG2b). As a result of immunodot blot analysis, the 4F7, 4H5, 5A5, 5A9, 6E4 and 6H4 antibodies were antibodies that did not recognize the Aβ monomer and bound specifically to the Aβ oligomer (see FIG. 14).

[Example 15] Inhibition ELISA
In order to examine the Aβ oligomer-selective binding ability of 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4), a solution prepared by mixing each antibody with serially diluted Aβ oligomer or Aβ monomer (inhibitor) was used. The reaction was added to the 96-well-immuno-plate that had been solidified with Aβ oligomer (see the method section). Commercially available 4G8 and 6E10 antibodies were used as control antibodies that bind to Aβ oligomers and Aβ monomers without distinction. When the antibody selectively binds to the Aβ oligomer, the antibody preliminarily mixed with the Aβ monomer is not absorbed by the Aβ monomer in the solution, and can bind to the solidified Aβ oligomer. On the contrary, since the antibody mixed in advance with the Aβ oligomer is absorbed by the Aβ oligomer in the solution, the amount of binding to the solidified Aβ oligomer decreases as the concentration increases. As a result, 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4) detected a concentration-dependent decrease in the amount of antibody binding with Aβ oligomers, whereas Aβ monomers had a binding capacity as large as Aβ oligomers. No decrease was detected (see FIG. 15). On the other hand, in 4G8 and 6E10, a similar concentration-dependent decrease in the amount of antibody binding was detected in Aβ monomer and Aβ oligomer (see FIG. 15). These results suggest that 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4) are antibodies that selectively bind to Aβ oligomers.

[Example 16] Aβ-induced neurotoxic neutralizing activity of 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4)
To evaluate whether 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4) have activity to neutralize Aβ-induced neurotoxicity, human neuroblastoma cells (SH-SY5Y) are used as antibodies The cells were cultured in a medium containing Aβ1-42 (12.5 μM) with or without addition for 24 hours, and the change in cytotoxicity caused by Aβ1-42 was measured. As a result, an increase in cytotoxicity was observed with the addition of the antibody in Control IgG (3F1). 4F7 and 4H5 also showed an increase in cytotoxicity due to antibody addition, but the rate was lower than that of 3F1 (see FIG. 16). With the remaining four types of antibodies (5A5, 5A9, 6E4, 6H4), a significant decrease in cytotoxicity was observed (see FIG. 16). From the above, four types of antibodies (5A5, 5A9, 6E4, 6H4) had strong activity to neutralize Aβ-induced neurotoxicity. On the other hand, 4F7 and 4H5 were presumed to have activity to neutralize Aβ-induced neurotoxicity because cytotoxicity was alleviated as compared with control IgG.

[Example 17] Aβ amyloid fibril formation inhibitory activity of 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4)
In order to examine whether 6 types of antibodies (4F7, 4H5, 5A5, 5A9, 6E4, 6H4) have Aβ amyloid fibril formation inhibitory activity, in the same solution composition as in Aβ-induced neurotoxicity experiment (in medium) Aβ amyloid fibril formation was measured by ThT fluorescence intensity measurement (see method section). In 6E4 and 6H4, antibody concentration-dependent suppression of fibril formation was observed (see FIG. 17). Since the other 4 antibodies (4F7, 4H5, 5A5, 5A9) also showed a tendency to suppress fiber as compared with control IgG, it is presumed that they have activity to suppress fibrosis.

[Discussion]
Our data show that monoclonal 1A9 or 2C3 is an antibody that specifically recognizes the “neurotoxic epitope” and “polymerizable epitope” of soluble Aβ polymer that causes toxic or antigenic fibrogenic activity. It is reported. Since monoclonal 1A9 or 2C3 does not react with a soluble Aβ monomer that is a physiological molecule, it can be concluded that a three-dimensional structure having an epitope recognized by 1A9 or 2C3 is characteristic of a soluble oligomeric polymer. Ultrafiltration and molecular sieve experiments revealed that the size of 1A9 or 2C3 immunoreactive oligomers exceeded 100 kDa (> 20-mer). From the results of morphological observation by AFM, it was also confirmed that the toxic polymer takes a heterogeneous form (granular, beaded, ring-shaped).

  In order to demonstrate that such toxic polymers are actually bioactive molecules with synaptic toxicity in vivo, anti-Aβ oligomer passive immunotherapy targeting 1A9 or 2C3 toxic polymers may be used prior to the onset of memory impairment. Started with young Tg2576 mice. We have presented for the first time evidence that passive immunization with anti-Aβ oligomer specific antibodies (1A9 and 2C3) protects against age-dependent memory loss that normally develops in Tg2576 mice. Impaired short-term memory as assessed by the Y-maze test herein is similar to that seen with mild cognitive impairment (MCI) and Aβ accumulation in early AD. In the Y-maze test, much better and almost normal results were observed in each of the Tg2576 mice treated with 1A9 and 2C3. For long-term memory as assessed by the novel object recognition task, the Morris water maze test or the conditioned fear task, both anti-Aβ oligomer antibodies kept performance nearly normal.

  Consistent with these antibodies' memory disorder prevention effect (memory maintenance effect), the antibody-treated mouse group showed a selective increase in A11 positive oligomers in the blood compared to the PBS-treated group. The 1A9 antibody treatment also showed an inhibitory effect on Aβ accumulation in the brain. In the 2C3 antibody treatment, the presence of A11 positive oligomers in the blood was proved more than the 1A9 antibody treatment, but the effect of suppressing Aβ accumulation in the brain was not clear. Therefore, the contribution to brain Aβ accumulation was considered to be greater for 1A9 oligomers than for 2C3 antibodies. The neurotoxic 1A9 polymer is a slightly sterically stabilized soluble toxic oligomer, and the neurotoxic 2C3 polymer is an extremely unstable and prone to conformational change that appears in the early stages of the polymerization process. Considering a sex intermediate, it is possible to explain its involvement in brain Aβ accumulation.

  In any case, the in vivo preventive effect of Alzheimer's disease presented by the anti-oligomer antibodies presented herein is the first direct evidence that toxic Aβ oligomers generated in vivo can interfere with neuronal function and lead to symptoms of Alzheimer's disease. Prove that.

  Our data also provide the first evidence for in vivo neurotoxicity of Aβ in the human brain. The human entorhinal cortex is well known to be susceptible to AD, but NFT formation and neuronal loss precede senile plaque formation, an exception that does not apply to the widely accepted amyloid cascade hypothesis The site has long been neglected.

  The present inventors have established and verified the hypothesis that invisible Aβ oligomers that have not been identified so far are harmful to neurons in the entorhinal cortex and cause memory impairment. To test this hypothesis, we performed semi-quantitative analysis of 1A9 or 2C3 immunoreactive 12-mer mainly in the entorhinal cortex of elderly humans at Braak NFT stages I-III. 1A9 or 2C3 immunoreactive 12-mer was already present in the entorhinal cortex of healthy individuals of Braak NFT stage I-II, increased as the Braak NFT stage progressed, and a significant increase was observed in AD. Thus, it was revealed in the human brain that the appearance of 1A9 or 2C3 immunoreactive 12-mer precedes the onset of cognitive dysfunction. On the other hand, the presence of 1A9 or 2C3 immunoreactive Aβ oligomers in senile plaques itself has been confirmed by biochemical and immunohistochemical methods, and insoluble amyloid fibrils themselves have neurotoxic neutralizing activity From these results, it was considered that in the situation where senile plaques were not formed despite the presence of such Aβ oligomers, Aβ oligomers themselves exerted in vivo toxicity and contributed to the development of memory impairment.

  Our data is the first report directly demonstrating memory impairment due to synaptic dysfunction mediated by endogenous Aβ oligomers in vivo, as described above. Furthermore, although active immunization [Janus D, 2000, Nature; Morgan D, 2000, Nature] or passive immunization [Bard F, 2222, Nat med; DeMattos RB, PNAS, 2001] has been performed, learning disorders And the current situation is that there is no speculation about the mechanism of how memory impairment is prevented. One widely proposed possibility is that the antibody reaches the brain through the blood brain barrier and directly neutralizes soluble Aβ oligomers that cause memory loss in vivo. A second possibility is the “sink theory” that the antibody itself acts in the periphery, depleting the Aβ peripheral pool in the blood and activating Aβ clearance from the brain. DeMattos et al. Reported that peripherally administered anti-Aβ antibodies rapidly extract not only brain Aβ monomers but also Aβ dimers into the plasma and rapidly extract brain Aβ into the CSF [DeMattos] RB et al, PNAS, 98; 8850-8855, 2001]. The present inventors also revealed that Aβ oligomers are present in human CSF and are increased in AD patients, confirming their usefulness as AD diagnostic markers. In addition, Aβ oligomers are present in the plasma of Tg2576 mice, and in passive immunotherapy by specific capture and neutralization by intravenous injection, it is not necessary to deliver antibodies into the brain, at the peripheral action point in the blood, For the first time, the evidence that it is possible to promote clearance of Aβ oligomers from the brain into the blood is also presented. Furthermore, we were able to present for the first time the evidence that senile plaque amyloid formation itself and indirect nerve cell injury (tumorous neurite formation) suppression via senile plaque amyloid were possible with passive immunotherapy. These results confirm that Aβ oligomers are the molecular basis of the onset of Alzheimer's disease, and that selective control by specific antibodies enables control of Alzheimer's disease itself from a preventive standpoint that is one step ahead of treatment. is there. Furthermore, it was proved that some of the administered antibodies migrated into the brain, and the action of directly neutralizing soluble Aβ oligomers in the brain and neonatal Fc-receptor [Deane R, 2005 , J Neurosci], the action of being transported into the blood, and the sink action, etc., acted in a complex manner, suggesting that the memory impairment inhibitory effect was exerted.

  In addition, an essential proposition for constructing such preventive treatment is the realization of a reliable pre-onset diagnosis of cases at high risk of developing AD. The significant increase in the CSF O / M ratio in AD reported herein was considered a strong candidate for achieving this goal.

  The antibody provided by the present invention can be used, for example, for intravenous preventive passive immunotherapy of Alzheimer's disease, biological markers (pre-onset diagnosis, disease observation, drug efficacy monitoring / determination) and the like.

  Further, the antibody provided by the present invention is expected to make a great contribution to the establishment of Alzheimer's disease state-causing responsible molecule selective prevention / treatment method and early diagnosis marker. Even in antibody therapies that should target intracerebral pathology, peripheral venous therapy is not enough to consider the intracerebral transfer, and peripheral venous therapy can be performed without considering intracerebral transfer. As a result of obtaining evidence that a part of the antibody moves into the brain and an additional effect due to its direct effect is obtained, it is considered that antibody therapy for Alzheimer's disease accelerates at a stretch.

Claims (18)

The antibody according to (1) or (2) below, which binds to an Aβ oligomer .
(1) The H chain having the amino acid sequence of SEQ ID NO: 89 as CDR1, the amino acid sequence of SEQ ID NO: 91 as CDR2, and the amino acid sequence of SEQ ID NO: 93 as CDR3 and SEQ ID NO: 95 as CDR1 An antibody comprising an L chain having the amino acid sequence of SEQ ID NO: 97 as CDR2 and the L chain having the amino acid sequence of SEQ ID NO: 99 as CDR3 (2) the amino acid of SEQ ID NO: 85 as VH A heavy chain having the sequence and
An antibody comprising an L chain having the amino acid sequence set forth in SEQ ID NO: 87 as VL   The antibody according to claim 1, characterized in that the antibody is a chimeric antibody or a humanized antibody.   A composition comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier.   An anticognitive dysfunction agent comprising the antibody according to claim 1 or the composition according to claim 3 as an active ingredient.   A therapeutic agent for Alzheimer's disease comprising the antibody according to any one of claims 1 to 2 or the composition according to claim 3 as an active ingredient.   An Alzheimer's disease progression inhibitor containing the antibody according to claim 1 or the composition according to claim 3 as an active ingredient.   A senile plaque formation inhibitor comprising the antibody according to claim 1 or the composition according to claim 3 as an active ingredient.   The A (beta) accumulation inhibitor which contains the antibody in any one of Claims 1-2, or the composition of Claim 3 as an active ingredient.   The anti-neurotoxic agent which contains the antibody in any one of Claims 1-2, or the composition of Claim 3 as an active ingredient.   An Aβ amyloid fibril formation inhibitor comprising the antibody according to claim 1 or the composition according to claim 3 as an active ingredient.   An anti-synaptic toxic agent containing the antibody according to claim 1 or the composition according to claim 3 as an active ingredient.   The method to measure A (beta) oligomer including the process of detecting the A (beta) oligomer contained in the sample extract | collected from the test subject using the antibody in any one of Claims 1-2.   Data useful for diagnosing whether or not a subject is a candidate for Alzheimer's disease, wherein the antibody according to any one of claims 1 to 2 is used to detect Aβ oligomers in a sample collected from the subject. How to provide. A method for providing data useful for diagnosis of whether or not a subject is a candidate for Alzheimer's disease, comprising the following steps, wherein the subject is higher when the amount described in step (b) is higher than that of a healthy subject: A method shown to be a candidate for Alzheimer's disease.
(A) a step of contacting a sample collected from a subject and the antibody according to any one of claims 1 to 2, and (b) a step of measuring the amount of Aβ oligomer in the sample. A method for providing data useful for diagnosis of whether or not a subject is a candidate for Alzheimer's disease, comprising the following steps, wherein the subject is higher when the ratio described in step (b) is higher than that of a healthy subject: A method shown to be a candidate for Alzheimer's disease.
(A) a step of contacting a sample collected from a subject, the antibody according to any one of claims 1 and 2 and an antibody that binds to Aβ monomer; (b) measuring a ratio of Aβ oligomer to Aβ monomer in the sample; Process   The method according to any one of claims 12 to 15, wherein the sample is blood or cerebrospinal fluid. The pharmaceutical containing the antibody in any one of Claims 1-2 for using for the method in any one of Claims 12-15. The kit containing the antibody in any one of Claims 1-2 for using for the method in any one of Claims 12-15.

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